TC-83-derived alphavirus vectors, particles and methods

ABSTRACT

The present disclosure provides TC-83 VEE-derived replicons, alphaviral replicon particles and immunogenic compositions containing TC-83 alphaviral replicon particles which direct the expression of at least one antigen when introduced into a suitable host cell. The TC-83 VEE-derived ARPs described herein are improved in that they are subject to a lower vector-specific immune response than prior art ARPs.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/132,711, filed May 18, 2005, which claims benefit of U.S. ProvisionalApplication No. 60/572,212, filed May 18, 2004, which application isincorporated by reference herein to the extent there is no inconsistencywith the present disclosure.

ACKNOWLEDGMENT OF FEDERAL RESEARCH SUPPORT

This invention was made, at least in part, through funding from theUnited States government, through grants from the National Institutes ofHealth, grant numbers 1U01 Al056438-01 and 5U01 Al 55071-02. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

The present invention relates to recombinant DNA technology, and inparticular to introducing foreign nucleic acid in a eukaryotic cell, andmore particularly to compositions and methods for producing alphavirusreplicon particles useful in immunotherapies and/or gene therapyapplications. In particular, the present invention discloses a geneticbackground for the alphavirus replicon particle system that is based onthe Venezuelan Equine Encephalitis virus (VEE) vaccine strain, TC-83.

A variety of viruses is included in the alphavirus genus, which is amember of the Togaviridae family. The alphaviral genome is asingle-stranded, messenger-sense RNA, modified at the 5′-end with amethylated cap and at the 3′-end with a variable-length poly (A) tract.Structural subunits containing a single viral protein, capsid, associatewith the RNA genome in an icosahedral nucleocapsid. In the virion, thenucleocapsid is surrounded by a lipid envelope covered with a regulararray of transmembrane protein spikes, each of which consists of threeheterodimeric complexes of two glycoproteins, E1 and E2. See Paredes etal., (1993) Proc. Natl. Acad. Sci. USA 90:9095-9099. The Sindbis andSemliki Forest viruses are considered the prototypical alphaviruses andhave been studied extensively. See Schlesinger, The Togaviridae andFlaviviridae, Plenum Publishing Corp., New York (1986). The VEE virushas also been studied extensively, see, e.g., U.S. Pat. Nos. 5,185,440,5,505,947, and 5,643,736.

The use of propagation-defective alphavirus particles, termed alphaviralreplicon particles, has shown great promise as a viral vector deliverysystem. Replicons are constructed to carry one or more heterologousantigens in place of some or all of the alphavirus structural genes. Thereplicons are introduced into alphavirus-permissive cells along with ahelper construct(s) that expresses the viral structural protein(s) notencoded by the replicon or, alternatively, the replicon is introducedinto a packaging cell capable of expressing the structural proteins. Thereplicon is then packaged, analogous to the packaging of the intactalphaviral genome, by the expressed structural proteins. These packagedreplicons, or alphaviral replicon particles, are then inoculated into ananimal. The particles enter the host cell, and the replicons thenexpress the introduced heterologous coding or other functionalsequence(s) at very high levels from the subgenomic mRNA. Subsequentviral progeny are prevented from assembly since the replicons do notencode all of the essential viral packaging (structural) genes.

Both the alphaviral genetic background for the replicon and thealphaviral structural proteins used to package the replicon have asignificant impact on the ultimate performance of the repliconparticles. The VEE virus has been preferred as a vaccine vector amongthe alphaviruses because it is naturally lymphotrophic, which leads tostrong cellular and humoral immune responses at relatively lowimmunization doses (Davis, N L et al. (1996) J. Virol. 70(6): 3781-7;MacDonald, G H and Johnston R E, (2000) J. Virol. 74(2): 914-922; CaleyI J et al. (1997) J. Virol. 71: 3031-3038; Hevey M et al. (1998)Virology 251(1): 28-37; Caley I J et al. (1999) Vaccine 17:23-24;Pushko, P et al. (2001) Vaccine 19:142-153).

Several strains of the Venezuelan Equine Encephalitis virus (VEE) areknown, and within those strains, subtypes have been recognized. VirulentVEE strains have been isolated during mosquito-borne epidemicencephalomyelitis in equids in tropical and sub-tropical areas of theNew World. One of the most virulent epizootic strains, the TrinidadDonkey (TRD) strain, which is in subtype IA/B, was passaged serially intissue culture to create a live, attenuated strain (Berge et al. (1961)Amer. J. Hyg. 73:209-218) known as TC-83. This strain elicitsVEE-specific neutralizing antibodies in most humans and equines and hasbeen used successfully as a vaccine in both species (McKinney et al.(1972) “Inactivated and live VEE vaccines—A Review, in VenezuelanEncephalitis, pp. 369-376, Sc. Pub. No. 243 Pan American HealthOrganization, Washington, D.C.; Walton T E et al. (1972) Am. J.Epidemiol. 95:247-254; Pittman P R et al. (1996) Vaccine 14(4):337-343). Nonetheless, this vaccine presents several problems in termsof safety and efficacy. First, it can cause adverse, sometimesmoderately severe reactions in human vaccines. Second, the TC-83 strainshows residual murine virulence and is lethal for suckling mice afterintracerebral (i.c.) or subcutaneous (s.c.) inoculation (Ludwig G et al.(2001) Am. J. Trop. Med. Hyg. January-February; 64(1-2):49-55). Third,the TC-83 strain has a significant percentage of non-responders inhumans, i.e., individuals who do not show a demonstrable humoralresponse after inoculation (Pittman P R et al. (1996) Vaccine 14(4):337-343). Finally, the TC-83 strain is known to be especially sensitiveto interferon, as compared to the parental TRD strain or other epizooticstrains of VEE (Spotts, D R et al. (1998) J. Virol. 72:10286-10291).Such enhanced sensitivity to interferon would lead one to expect thatthe heterologous genes in a replicon particle would be expressed lessefficiently in an infected cell and/or that such particles would be lessimmunogenic in vivo. All of these detrimental factors associated withthe TC-83 vaccine strain of VEE have led previous researchers to searchfor better attenuated strains to use as either propagation-competent VEEvectors or in replicon particle systems (e.g. Davis N L et al. (1994)Arch. Virol. Suppl. 9:99-109; Davis N L et al. (1996) J. Virol.70(6):3781; Pushko et al. (1997) Ibid.; Pratt W D et al. (2003) Vaccine21(25-26): 3854-3862).

There is a continuing need to optimize the combination of mutations andalphavirus strain to provide the most effective alphavirus repliconparticle for use in vaccine and/or gene therapy applications.

SUMMARY OF THE INVENTION

The present invention provides compositions of infective,replication-defective, highly immunogenic alphavirus replicon particlesbased on a particular alphavirus strain, i.e., the TC-83 of VEE, andmethods of preparation thereof. As described previously (see, forexample, U.S. Pat. Nos. 5,792,462; 6,156,558; 5,811,407; 6,008,035;6,583,121; WO 03/023026; U.S. Publication No. 2003/0119182, allincorporated herein by reference), functional alphavirus repliconparticles have been made from several different alphaviruses andchimeras thereof (see, for example, U.S. Publication No 2003/0148262).These particles are useful in vaccine and gene therapy applications, andthe optimal characteristics of the alphavirus replicon particles differin these applications. For instance, it may be useful to reduce theexpression of proteins from the replicon during gene therapyapplications, and thus techniques have been developed in the art toreduce such expression (see e.g. U.S. Pat. Nos. 5,843,723 and6,451,592). In the case of vaccine applications, maximizing theexpression of the heterologous RNA from the replicon, minimizing anyanti-vector responses, and targeting the tissues and cells of the immunesystem are desirable features. The alphaviruses Venezuelan EquineEncephalitis (VEE) virus and South African Arbovirus No. 86 have provedparticularly useful in the vaccine applications. To improve the safetyof these alphavirus vectors in the rare event that areplication-competent virus is generated, at least one attenuatingmutation has been introduced into the alphaviral genomic fragments. Thepresent inventors have now discovered that the TC-83 strain of VEE canbe used as the genetic background for an alphavirus replicon particlesystem which provides a surprisingly effective VEE particle preparationfor use in immunogenic compositions and which has other surprisinglyadvantageous properties useful in a vaccine vector system, including theability to prepare purified preparations with ease.

The present inventors have discovered that the TC-83 strain of VEE is asurprisingly good alphavirus strain from which to derive a repliconvaccine particle. A complete sequence of the TC-83 sequence waspublished (Kinney R M et al. (1989) Virology 170:19-30; with correctionnoted in Kinney R M et al. (1993) J. Virol. 67(3):1269-1277). The genomeof this live, attenuated vaccine strain carries 12 differences from thevirulent, parental strain from which it was derived. These mutationsare: a single nucleotide substitution (G→A) at nucleotide 3 of the 5′non coding region; amino acid substitutions at nsP2-16 (Ala→Asp),nsP3-260 (Ser→Thr), E2-7 (Lys→Asn), E2-85 (His→Tyr), E2-120 (Thr→Arg),E2-192 (Val→Asp), E2-296 (Thr→Ile), and E1-161 (Leu→Ile); 2 silentnucleotide substitutions at E2-278 (U→C) and E1-211 (A→U), and a singlenucleotide deletion at nucleotide 11,405 in the 3′ non-coding region(UU→U). Kinney et al. 1993 Ibid. have suggested that the attenuatedphenotype of the live TC-83 strain (i.e. reduced neurovirulence in mice)is due to the nucleotide 3 mutation (G to A) and the E2 mutations,particularly the E2-120 mutation. It has been shown that this nucleotide3 mutation, when introduced into a wild-type strain of VEE, attenuatesthe strain (White L J et al. (2001) J. Virol 75: 3706-3718). However,the methods used do not exclude contributions from other mutations, andthe existence of the numerous other nonconservative mutations in theTC-83 genome make it impossible to predict whether it can serve as aneffective genetic background for the replicon particle system.

The inventors have now produced a replicon particle vaccine based on theTC-83 strain, and it has several surprisingly advantageouscharacteristics for both vaccine and gene therapy applicationsincluding, but not limited to, much higher yields as compared to thoseachieved with particles based on wild-type VEE or on those carryingother attenuating mutations; lowered anti-vector responses; increasedpurity; excellent immunogenicity that is comparable to other VEE strainscarrying only one, two or three attenuating mutations, and nonon-responsiveness, in contrast to the noted non-responsiveness ofanimals to the live TC-83 strain used as a vaccine.

Additionally, the inventors have discovered that packaging an alphavirusreplicon in the VEETC83 structural proteins results in significantlyhigher yields of replicon particle vaccines from cell cultures. Thus,the VEETC83 structural proteins can be advantageously used to packagereplicons from other alphaviruses, including other strains of VEE.

Thus, the present invention provides a recombinant alphavirus particlecomprising (i) an alphavirus replicon RNA encoding one or moreheterologous RNA sequences, wherein the replicon RNA comprises a 5′sequence which initiates transcription of alphavirus RNA, one or morenucleotide sequences which together encode those TC-83 alphavirusnonstructural proteins necessary for replication of the replicon RNA, ameans for expressing the polypeptide encoded by the heterologous RNA(s),and a 3′ RNA polymerase recognition sequence, (ii) a TC-83 derivedcapsid protein; and (iii) alphavirus glycoproteins derived from TC-83.

The present invention also provides other VEE vaccine strains,especially those with characteristics similar to those of TC-83, whichcan be engineered for use in immunogenic replicon particle compositions.

Also provided is a population of infectious, propagation-defective,alphavirus particles, wherein the population comprises repliconparticles comprising a VEE TC-83 replicon RNA comprising an alphaviruspackaging signal, one or more heterologous RNA sequence(s) encoding anucleic acid of interest and lacking sequences encoding alphavirusstructural proteins, and wherein the population contains no more than 10replication-competent TC-83 viral particles per 10⁸ TC-83 repliconparticles.

Also provided is a composition comprising a population of infectious,propagation-defective, alphavirus particles, wherein (1) each particlecomprises an alphavirus replicon RNA encoding one or more heterologousRNA sequences and lacks sequences encoding any alphavirus structuralproteins, (2) the population has no detectable replication competentviruses (RCV), as measured by passage on cell cultures, (3) the repliconRNA is derived from TC-83, and wherein the population is formulated witha pharmaceutically acceptable carrier. The alphavirus structuralproteins can be derived from the alphavirus VEE vaccine strain TC-83, awild-type VEE strain, or other strains of VEE containing one or moreattenuating mutations in the alphaviral genomic sequences encoding thestructural proteins. In a specific embodiment, the TC-83 structuralproteins may have one or more additional attenuating mutationsintroduced, e.g. at E1-81 (e.g. from Phe to Ile).

Also provided is a composition comprising a population of infectious,propagation-defective, alphavirus particles, wherein (1) each particlecomprises an alphavirus replicon RNA encoding one or more heterologousRNA sequences and lacks sequences encoding any alphavirus structuralproteins, (2) the structural proteins comprising the coat of theparticles are derived from VEETC83, and (3) the population has nodetectable replication competent viruses (RCV), as measured by passageon cell cultures, and wherein the population is formulated with apharmaceutically acceptable carrier. In this composition, the alphavirusreplicon RNA is derived from a wild-type VEE strain or other non-TC83strains of VEE containing one or more attenuating mutations in thealphaviral genomic sequences contained within the replicon. In aspecific embodiment, the TC-83 structural proteins may have one or moreadditional attenuating mutations introduced, e.g. at E1-81 (e.g. fromPhe to Ile).

Also provided is a method of producing an immune response in a subject,comprising administering to the subject an effective amount of animmunogenic composition comprising a population of infectious,propagation-defective alphavirus particles in apharmaceutically-acceptable carrier, wherein the composition comprisesparticles comprising a VEE TC-83 replicon RNA comprising an alphaviruspackaging signal, one or more heterologous RNA sequence(s) encoding animmunogen and lacking sequences encoding alphavirus structural proteins,and wherein the composition has less than 10 replication-competent TC-83particles per 10⁸ TC-83 replicon particles.

Also provided is a helper cell for producing an infectious,propagation-defective alphavirus particle comprising (1) a VEETC83replicon RNA comprising a heterologous RNA sequence, for example, acoding sequence heterologous to the virus, and lacking sequencesencoding alphavirus structural proteins; and (2) one or more nucleicacids encoding the TC-83 structural proteins. Alternatively thestructural proteins can be selected from the group consisting ofwild-type VEE structural glycoproteins, VEE 3014 structuralglycoproteins, VEE 3040 glycoproteins, VEE 3042 glycoproteins, and VEE3526 glycoproteins, but preferably from among VEE structuralglycoproteins which contain amino acid substitutions that conferattenuated virulence, and the VEE capsid is produced from the wild-typesequence or from a sequence in which the auto-proteolytic cleavage sitehas been deleted.

Also provided is a helper cell for producing an infectious,propagation-defective alphavirus particle comprising (1) an alphavirusreplicon RNA comprising a heterologous RNA sequence, for example, acoding sequence heterologous to the virus, and lacking sequencesencoding alphavirus structural proteins; and (2) one or more nucleicacids encoding the TC-83 structural proteins.

The present invention further provides a method of producing infectious,propagation-defective TC-83 replicon particles comprising introducinginto a population of cells a recombinant DNA molecule encoding all theVEE structural proteins, and a TC-83 replicon RNA encoding at least oneheterologous RNA, such that infectious, propagation-defective TC-83replicon particles are produced, and wherein the VEE structuralglycoproteins are derived from one of the following VEE strains: TC-83,3014, 3040, 3042 and 3526. These strains are referred to herein asVEETC83, VEE3014, etc.

Also provided is a method of producing infectious, propagation-defectivealphavirus replicon particles comprising introducing into a populationof cells a recombinant DNA molecule encoding all the VEETC83 structuralproteins, and an alphavirus replicon RNA encoding at least oneheterologous RNA, such that infectious, propagation-defective repliconparticles are produced, and wherein the VEE replicon RNA is derived froma wild-type VEE strain or incorporates at least one attenuatingmutations, such as the mutation to an A at nucleotide 3.

A method of producing infectious, propagation-defective alphavirusreplicon particles comprising introducing into a population of cells (i)two recombinant nucleic acid molecules, each of which encodes at leastone, but not all of VEE structural proteins and (ii) a TC-83 repliconRNA encoding at least one heterologous RNA, wherein the two recombinantnucleic acid molecules together encode all VEE structural proteinsrequired to produce infectious, propagation-defective TC-83 repliconparticles in the cells, and further wherein the alphaviral structuralproteins are derived from one of the following VEE strains: TC-83, 3014,3040, 3042 and 3526. These strains are typically referred to in thisapplication as “VEETC83”, “VEE3014,” etc.

Also provided is a method of providing advantageously purified,infectious, propagation-defective TC-83 replicon particles by heparinaffinity chromatography, either by column or batch purification methods.The unique heparin-binding characteristics of the TC-83 derived repliconparticles allow for removal of contaminating proteins and nucleic acidsthrough a single purification step.

Also provided are methods of eliciting an immune response in a subject,comprising administering to the subject an immunogenic amount of thepopulation of replicon particles of this invention.

The present invention is also applicable to the production of liveattenuated alphavirus vaccines, which may or may not carry heterologousgenes for expression in the vaccinee, as described in U.S. Pat. No.5,643,576, or live attenuated alphavirus vectors which direct theexpression of functional RNAs (such as antisense, suppressing RNAs orinterfering RNAs or RNAs which encode therapeutic proteins. The methodof the present invention comprises the steps of (a) introducing theTC-83 replicon nucleic acid into a host cell, wherein said repliconnucleic acid contains at least an alphavirus packaging signal and atleast one coding sequence for a protein or functional RNA of interestexpressible in said alphaviral replicon nucleic acid, wherein the hostcell is capable of expressing alphavirus structural proteins required toproduce ARPs, to produce a modified host cell; (b) culturing saidmodified host cell in a medium under conditions allowing expression ofthe structural proteins and replication of the alphaviral repliconnucleic acid, and then packaging of the alphaviral replicon nucleic acidto form ARPs; (c), optionally separating the modified host cells fromthe medium, and (d) after step (b) or (c) contacting the modified hostcells with an aqueous solution having an ionic strength of at leastapproximately 0.20 M, desirably from about 0.5 to about 5 M, (herein the“Release Medium”) to release the ARPs into the aqueous solution toproduce an ARP-containing solution. The ionic strength of the ReleaseMedium can be achieved using salts which do not inactivate the virionsor ARPs, and suitable salts include, but are not limited to, sodiumchloride, magnesium chloride, ammonium chloride, ammonium acetate,potassium chloride, calcium chloride, ammonium bicarbonate, and heparinFast Flow. Desirably the Release Medium comprises a buffer with a pHfrom about 6 to about 9, preferably from about 6.5 to about 8.5. Wherethe cells are not separated from the medium, the ionic strength of themedium can be raised by the addition of solid salts or a concentratedsolution to provide the increased ionic strength for releasing the ARPs(or virions) from the cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the elution profile of TC-83 virus replicon particlesduring heparin affinity chromatography.

FIG. 2 shows the results of SDS-PAGE of TC-83 virus replicon particlesafter heparin affinity chromatography, with the proteins visualized bysilver staining and by Western blotting using capsid-specific andglycoprotein-specific antibodies and staining.

FIG. 3 is a plasmid map of the TC-83 replicon cloning vector pVEK.

DETAILED DESCRIPTION OF THE INVENTION

The following discussion and definitions are provided to improve theclarity of the present disclosure to one of ordinary skill in therelevant art.

In the context of the present application, nm means nanometer, ml meansmilliliter, VEE means Venezuelan Equine Encephalitis virus, EMC meansEncephalomyocarditis virus, BHK means baby hamster kidney cells, HAmeans hemagglutinin gene, GFP means green fluorescent protein gene, Nmeans nucleocapsid, FACS means fluorescence activated cell sorter, IRESmeans internal ribosome entry site, and FBS means Fetal Bovine Serum.The expression “E2 amino acid (e.g., Lys, Thr, etc.) number” indicatesdesignated amino acid at the designated residue of the E2 gene, and isalso used to refer to amino acids at specific residues in the E1 gene.

As used herein, the term “alphavirus” has its conventional meaning inthe art, and includes the various species such as VEE, SFV, Sindbis,Ross River Virus, Western Equine Encephalitis Virus, Eastern EquineEncephalitis Virus, Chikungunya, S.A. AR86, Everglades virus, Mucambo,Barmah Forest Virus, Middelburg Virus, Pixuna Virus, O'nyong-nyongVirus, Getah Virus, Sagiyama Virus, Bebaru Virus, Mayaro Virus, UnaVirus, Aura Virus, Whataroa Virus, Banbanki Virus, Kyzylagach Virus,Highlands J Virus, Fort Morgan Virus, Ndumu Virus, and Buggy CreekVirus. The preferred alphaviruses used in the constructs and methods ofthe claimed invention are VEE, S.A. AR86, Sindbis (e.g. TR339, see U.S.Pat. No. 6,008,035), and SFV.

“Alphavirus-permissive cells” employed in the methods of the presentinvention are cells that, upon transfection with a complete viral RNAtranscript, are capable of producing viral particles. Alphaviruses havea broad host range. Examples of suitable packaging cells include, butare not limited to, Vero cells, baby hamster kidney (BHK) cells, chickenembryo fibroblast cells, DF-1, 293, 293T, Chinese Hamster Ovary (CHO)cells, and insect cells.

As used herein, the phrases “attenuating mutation” and “attenuatingamino acid,” mean a nucleotide mutation (which may or may not be in aregion of the viral genome encoding polypeptides) or an amino acid codedfor by a nucleotide mutation, which in the context of a live virus,result in a decreased probability of the alphavirus causing disease inits host (i.e., a loss of virulence), in accordance with standardterminology in the art, See, e.g., B. Davis, et al., Microbiology 132(3d ed. 1980), whether the mutation be a substitution mutation, or anin-frame deletion or addition mutation. The phrase “attenuatingmutation” excludes mutations which would be lethal to the virus, unlesssuch a mutation is used in combination with a “restoring” mutation whichrenders the virus viable, albeit attenuated. Exemplary attenuatingmutations in VEE structural proteins include, but are not limited to,those described in U.S. Pat. No. 5,505,947 to Johnston et al., U.S. Pat.No. 5,185,440 to Johnston et al., U.S. Pat. No. 5,643,576 to Davis etal., U.S. Pat. No. 5,792,462 to Johnston et al., and U.S. Pat. No.5,639,650 to Johnston et al., the disclosures of which are incorporatedherein in their entireties by reference. Specific attenuating mutationsfor the VEE E1 glycoprotein include an attenuating mutation at any oneof amino acid positions 81, 272 or 253. Alphavirus replicon particlesmade from the VEE-3042mutant contain an isoleucine substitution atE1-81, (amino acid 81 of the E1 protein) and virus replicon particlesmade from the VEE-3040 mutant contain an attenuating mutation at E1-253.Specific attenuating mutations for the VEE E2 glycoprotein include anattenuating mutation at any one of amino acid positions 76, 120 , or209. Alphavirus replicon particles made from the VEE-3014 mutant containattenuating mutations at both E1-272 and at E2-209 (see U.S. Pat. No.5,792,462). A specific attenuating mutation for the VEE E3 glycoproteinincludes an attenuating mutation consisting of a deletion of amino acids56-59. Virus replicon particles made from the VEE-3526 mutant containthis deletion in E3 (aa56-59) as well as a second attenuating mutationat E1-253. For alphaviruses generally, deletion or substitutionmutations in the cleavage domain between E3 and E2, which result in theE3/E2 polyprotein not being cleaved, are attenuating.

The terms “5′ alphavirus replication recognition sequence” and “3′alphavirus replication recognition sequence” refer to the sequencesfound in alphaviruses, or sequences derived therefrom, that arerecognized by the nonstructural alphavirus replicase proteins and leadto replication of viral RNA. These are sometimes referred to as the 5′and 3′ ends, or alphavirus 5′ and 3′ sequences. In the constructs ofthis invention, the use of these 5′ and 3′ ends will result inreplication of the RNA sequence encoded between the two ends. The 3′alphavirus replication recognition sequence as found in the alphavirusis typically approximately 300 nucleotides in length, which contains amore well defined, minimal 3′ replication recognition sequence. Theminimal 3′ replication recognition sequence, conserved amongalphaviruses, is a 19 nucleotide sequence (Hill et al., J. Virology,2693-2704, 1997). These sequences can be modified by standard molecularbiological techniques to further minimize the potential forrecombination or to introduce cloning sites, with the proviso that theymust be recognized by the alphavirus replication machinery.

The term “minimal 5′ alphavirus replication recognition sequence” refersto the minimal sequence that allows recognition by the nonstructuralproteins of the alphavirus but does not result in significantpackaging/recombination of RNA molecules containing the sequence. In apreferred embodiment, the minimal 5′ alphavirus replication recognitionsequence results in a fifty to one-hundred fold decrease in the observedfrequency of packaging/recombination of the RNA containing thatsequence. Packaging/recombination of helpers can be assessed by severalmethods, e.g. the method described by Lu and Silver (J. Virol. Methods2001, 91(1): 59-65).

The terms “alphavirus RNA replicon”, “alphavirus replicon RNA”,“alphavirus RNA vector replicon”, “replicon”, and “vector replicon RNA”are used interchangeably to refer to an RNA molecule expressingnonstructural protein genes such that it can direct its own replication(amplification) and comprises, at a minimum, 5′ and 3′ alphavirusreplication recognition sequences (which may be the minimal sequences,as defined above, but may alternatively be the entire regions from thealphavirus), coding sequences for alphavirus nonstructural proteins, anda polyadenylation tract. It may additionally contain a promoter and/oran IRES. Specific replicons useful in the claimed invention include: areplicon based on VEETC83, herein referred to as a “VEETC83 replicon”; areplicon based on the wild-type sequence of VEE, herein referred to as a“VEE3000 replicon”; and a replicon based on VEE3000 but additionallyincluding one of the attenuating mutations present in TC83, namely themutation to an “A” at nucleotide 3, herein referred to as “VEE3000nt3A”.

The alphavirus RNA vector replicon is designed to express a heterologousnucleic acid, e.g. a gene, of interest, also referred to herein as aheterologous RNA or heterologous sequence, which can be chosen from awide variety of sequences derived from viruses, prokaryotes oreukaryotes. Examples of categories of heterologous sequences include,but are not limited to, immunogens, including antigenic proteins,cytokines, toxins, therapeutic proteins, enzymes, antisense sequences,and immune response modulators.

The alphavirus RNA replicons of this invention may also be engineered toexpress alphavirus structural proteins, thereby generating a vaccineagainst the alphavirus(es) from which the structural proteins arederived. Johnston et al. and Polo et al. (cited in the background)describe numerous constructs for such alphavirus RNA replicons, and suchconstructs are incorporated herein by reference. Specific embodiments ofthe alphavirus RNA replicons utilized in the claimed invention maycontain one or more attenuating mutations, an attenuating mutation beinga nucleotide deletion, addition, or substitution of one or morenucleotide(s), or a mutation that comprises rearrangement or chimericconstruction which results in a loss of virulence in a live viruscontaining the mutation as compared to the appropriate wild-typealphavirus. Examples of an attenuating nucleotide substitution(resulting in an amino acid change in the replicon) include a mutationat nsP1 amino acid position 538, nsP2 amino acid position 96, or nsP2amino acid position 372 in the alphavirus S.A.AR86.

The terms “alphavirus structural protein/protein(s)” refers to one or acombination of the structural proteins encoded by alphaviruses. Theseare produced by the virus as a polyprotein and are represented generallyin the literature as C-E3-E2-6k-E1. E3 and 6k serve as membranetranslocation/transport signals for the two glycoproteins, E2 and E1.Thus, use of the term E1 herein can refer to E1, E3-E1, 6k-E1, orE3-6k-E1, and use of the term E2 herein can refer to E2, E3-E2, 6k-E2,or E3-6k-E2.

The term “helper(s)” or helper constructs refers to a nucleic acidmolecule that is capable of expressing one or more alphavirus structuralproteins.

The terms “helper cell” and “packaging cell” are used interchangeablyherein and refer to the cell in which alphavirus replicon particles areproduced. The helper cell comprises a set of helpers that encode one ormore alphavirus structural proteins. As disclosed herein, the helpersmay be RNA or DNA. The cell can be any cell that isalphavirus-permissive. In certain embodiments of the claimed invention,the helper or packaging cell may additionally include a heterologousRNA-dependent RNA polymerase and/or a sequence-specific protease.

The terms “alphavirus replicon particles”, “virus replicon particles”,“VRPs” or “recombinant alphavirus particles”, used interchangeablyherein, mean a virion-like structural complex incorporating analphavirus replicon RNA that expresses one or more heterologous RNAsequences. Typically, the virion-like structural complex includes one ormore alphavirus structural proteins embedded in a lipid envelopeenclosing a nucleocapsid comprised of capsid and replicon RNA. The lipidenvelope is typically derived from the plasma membrane of the cell inwhich the particles are produced. Preferably, the alphavirus repliconRNA is surrounded by a nucleocapsid structure comprised of thealphavirus capsid protein, and the alphavirus glycoproteins are embeddedin the cell-derived lipid envelope. These replicon particles arepropagation-defective (or synonymously “replication defective”), whichmeans that the particles produced in a given host cell cannot produceprogeny particles in the host cell, due to the absence of the helperfunction, i.e. the alphavirus structural proteins required for packagingthe replicon nucleic acid. However, the replicon nucleic acid is capableof replicating itself and being expressed within the host cell intowhich it has been introduced. Replicon particles of this invention maybe referred to as VEETC83 replicon particles, and this refers toparticles comprising either a TC83 replicon RNA or TC83 structuralproteins, or both a TC83 replicon RNA and TC83 structural proteins.

Any amino acids which occur in the amino acid sequences referred to inthe specification have their usual three- and one-letter abbreviationsroutinely used in the art: A, Ala, Alanine; C, Cys, Cysteine; D, Asp,Aspartic Acid; E, Glu, Glutamic Acid; F, Phe, Phenylalanine; G, Gly,Glycine; H, H is, Histidine; I, Ile, Isoleucine; K, Lys, Lysine; L, Leu,Leucine; M, Met, Methionine; N, Asn, Asparagine; P, Pro, Proline; Q,Gln, Glutamine; R, Arg, Arginine; S, Ser, Serine; T, Thr, Threonine; V,Val, Valine; W, Try, Tryptophan; Y, Tyr, Tyrosine.

As used herein, expression directed by a particular sequence is thetranscription of an associated downstream sequence, i.e. production ofmessenger RNA from a DNA molecule or production of messenger RNA from analphavirus subgenomic promoter. If appropriate and desired for theparticular application, the transcribed mRNA is then translated, i.e.protein is synthesized. Thus, in one embodiment of this invention, thereplicon or helper construct comprises a subgenomic promoter whichdirects transcription of a messenger RNA encoding the heterologousnucleic acid of interest (NOI) or the transcription of an mRNA encodingone or more alphavirus structural proteins, respectively. These mRNAsare “capped” within the eukaryotic cell, i.e. a methyl-7-guanosine(5′)pppN structure is present at the 5′ end of the mRNA (the “cap” or“5′ cap”), and this cap is recognized by the translation initiationfactors that synthesize protein from the mRNA. Thus, the 26S promoterdirects transcription, and the “cap” provides the initiation signal fortranslation.

In another embodiment, the replicon or helper construct comprises apromoter that directs transcription; an IRES element; and a codingsequence, and the IRES element is operably located such that translationof the coding sequence is via a cap-independent mechanism directed bythe IRES element, either in whole or in part, described in detail inWIPO Publication No. WO 2004/085660. In particular, control of nucleicacid expression at the level of translation is accomplished byintroducing an internal ribosome entry site (IRES) downstream of analphavirus 26S subgenomic promoter and upstream of the coding sequenceto be translated. The IRES element is positioned so that it directstranslation of the mRNA, thereby minimizing, limiting or preventinginitiation of translation of the mRNA from the 5′ cap. This“IRES-directed,” cap-independent translation does not require or resultin any significant modification of alphavirus non-structural proteingenes that could alter replication and transcription. In specificembodiments, the replicon and/or helper construct can comprise a spacernucleic acid located between the promoter and the IRES element. Thespacer nucleic acid can comprise or consist of any random or specificnon-coding nucleic acid sequence which is of a length sufficient toprevent at least some, and in some embodiments, all translation from the5′ cap of a messenger RNA, such that translation is then directed by theIRES, in part or in whole. Alternatively, the spacer nucleic acid can beof a length and sequence structure that imparts sufficient secondarystructure to the nucleic acid to prevent at least some and possibly alltranslation activity from the 5′ cap of a messenger RNA.

Suitable IRES elements include, but are not limited to, viral IRESelements from picornaviruses, e.g., poliovirus (PV) or the humanenterovirus 71, e.g. strains 7423/MS/87 and BrCr thereof; fromencephalomyocarditis virus (EMCV); from foot-and-mouth disease virus(FMDV); from flaviviruses, e.g., hepatitis C virus (HCV); frompestiviruses, e.g., classical swine fever virus (CSFV); fromretroviruses, e.g., murine leukemia virus (MLV); from lentiviruses,e.g., simian immunodeficiency virus (SIV); from cellular mRNA IRESelements such as those from translation initiation factors, e.g., eIF4Gor DAP5; from transcription factors, e.g., c-Myc (Yang and Sarnow,Nucleic Acids Research 25: 2800-2807 (1997)) or NF-κB-repressing factor(NRF); from growth factors, e.g., vascular endothelial growth factor(VEGF), fibroblast growth factor (FGF-2) and platelet-derived growthfactor B (PDGF B); from homeotic genes, e.g., Antennapedia; fromsurvival proteins, e.g., X-linked inhibitor of apoptosis (XIAP) orApaf-1; from chaperones, e.g., immunoglobulin heavy-chain bindingprotein BiP (Martinez-Salas et al., Journal of General Virology 82:973-984, (2001)), from plant viruses, as well as any other IRES elementsnow known or later identified.

In specific embodiments, the IRES element of this invention can bederived from, for example, encephalomyocarditis virus (EMCV, GenBankaccession # NC001479), cricket paralysis virus (GenBank accession #AF218039), Drosophila C virus (GenBank accession # AF014388), Plautiastali intestine virus (GenBank accession # AB006531), Rhopalosiphum padivirus (GenBank accession # AF022937), Himetobi P virus (GenBankaccession # AB017037), acute bee paralysis virus (GenBank accession #AF150629), Black queen cell virus (GenBank accession #AF183905),Triatoma virus (GenBank accession # AF178440), Acyrthosiphon pisum virus(GenBank accession # AF024514), infectious flacherie virus (GenBankaccession # AB000906), and/or Sacbrood virus (Genbank accession #AF092924). In addition, synthetic IRES elements have been described,which can be designed, according to methods know in the art to mimic thefunction of naturally occurring IRES elements (see Chappell, S A et al.Proc. Natl. Acad. Sci. USA (2000) 97(4):1536-41.

In specific embodiments, the IRES element can be an insect IRES elementor other non-mammalian IRES element that is functional in the particularhelper cell line chosen for packaging of the recombinant alphavirusparticles of this invention, but would not be functional, or would beminimally functional, in a target host cell for the particles (e.g. ahuman subject). This is useful for those NOIs which are either toxic tothe packaging cell or are detrimental to the alphavirus packagingprocess.

The phrases “structural protein” or “alphavirus structural protein” asused herein refer to one or more of the alphaviral-encoded proteinswhich are required for packaging of the RNA replicon, and typicallyinclude the capsid protein, E1 glycoprotein, and E2 glycoprotein in themature alphavirus (certain alphaviruses, such as Semliki Forest Virus,contain an additional protein, E3, in the mature coat). The term“alphavirus structural protein(s)” refers to one or a combination of thestructural proteins encoded by alphaviruses. These are synthesized (fromthe viral genome) as a polyprotein and are represented generally in theliterature as C-E3-E2-6k-E1. E3 and 6k serve as membranetranslocation/transport signals for the two glycoproteins, E2 and E1.Thus, use of the term E1 herein can refer to E1, E3-E1, 6k-E1, orE3-6k-E1, and use of the term E2 herein can refer to E2, E3-E2, 6k-E2,or E3-6k-E2.

As described herein, the nucleic acid sequences encoding structuralproteins of the alphavirus are distributed among one or more helpernucleic acid molecules (e.g., a first helper RNA (or DNA) and a secondhelper RNA (or DNA)). In addition, one or more structural proteins maybe located on the same molecule as the replicon nucleic acid, providedthat at least one structural protein is deleted from the replicon RNAsuch that the replicon and resulting alphavirus particle are propagationdefective with respect to the production of further alphavirusparticles. As used herein, the terms “deleted” or “deletion” mean eithertotal deletion of the specified segment or the deletion of a sufficientportion of the specified segment to render the segment inoperative ornonfunctional, in accordance with standard usage. See, e.g., U.S. Pat.No. 4,650,764 to Temin et al. Distribution of the helper nucleic acidsequences among multiple nucleic acid molecules minimizes the frequencyat which replication competent virus (RCV) are generated throughrecombination events. In the case of the DNA helper constructs that donot employ alphaviral recognition signals for replication andtranscription, the theoretical frequency of recombination is lower thanthe bipartite RNA helper systems that employ such signals.

The helper cell, also referred to as a packaging cell, used to producethe infectious, propagation defective alphavirus particles, must expressor be capable of expressing alphavirus structural proteins sufficient topackage the replicon nucleic acid. The structural proteins can beproduced from a set of RNAs, typically two, that are introduced into thehelper cell concomitantly with or prior to introduction of the repliconvector. The first helper RNA includes RNA encoding at least onealphavirus structural protein but does not encode all alphavirusstructural proteins. The first helper RNA may comprise RNA encoding thealphavirus E1 glycoprotein, but not encoding the alphavirus capsidprotein and the alphavirus E2 glycoprotein. Alternatively, the firsthelper RNA may comprise RNA encoding the alphavirus E2 glycoprotein, butnot encoding the alphavirus capsid protein and the alphavirus E1glycoprotein. In a further embodiment, the first helper RNA may compriseRNA encoding the alphavirus E1 glycoprotein and the alphavirus E2glycoprotein, but not the alphavirus capsid protein. In a fourthembodiment, the first helper RNA may comprise RNA encoding thealphavirus capsid, but none of the alphavirus glycoproteins. In a fifthembodiment, the first helper RNA may comprise RNA encoding the capsidand one of the glycoproteins, i.e. either E1 or E2, but not both.

In preferred embodiments employing two helper RNAs, in combination withany one of these first helper RNAs, the second helper RNA encodes theone or more alphavirus structural proteins not encoded by the firsthelper RNA. For example, where the first helper RNA encodes only thealphavirus E1 glycoprotein, the second helper RNA encodes both thealphavirus capsid protein and the alphavirus E2 glycoprotein. Where thefirst helper RNA encodes only the alphavirus capsid protein, the secondhelper RNA encodes both the alphavirus glycoproteins. Where the firsthelper RNA encodes only the alphavirus E2 glycoprotein, the secondhelper RNA encodes both the alphavirus capsid protein and the alphavirusE1 glycoprotein. Where the first helper RNA encodes both the capsid andalphavirus E1 glycoprotein, the second helper RNA may include RNAencoding one or both of the alphavirus capsid protein and the alphavirusE2 glycoprotein.

In all of the helper nucleic acids, it is understood that thesemolecules further comprise sequences necessary for expression(encompassing translation and where appropriate, transcription orreplication signals) of the encoded structural protein sequences in thehelper cells. Such sequences can include, for example, promoters (eitherviral, prokaryotic or eukaryotic, inducible or constitutive), IRESes,and 5′ and 3′ viral replicase recognition sequences. In the case of thehelper nucleic acids expressing one or more glycoproteins, it isunderstood from the art that these sequences are advantageouslyexpressed with a leader or signal sequence at the N-terminus of thestructural protein coding region in the nucleic acid constructs. Theleader or signal sequence can be derived from the alphavirus, forexample E3 or 6k, or it can be a heterologous sequence such as a tissueplasminogen activator signal peptide or a synthetic sequence. Thus, asan example, a first helper nucleic acid may be an RNA molecule encodingcapsid-E3-E1, and the second helper nucleic acid may be an RNA moleculeencoding capsid-E3-E2. Alternatively, the first helper RNA can encodecapsid alone, and the second helper RNA can encode E3-E2-6k-E1.Additionally, the packaging signal(s) or “encapsidation sequence(s)”that are present in the viral genome are not present in all of thehelper nucleic acids. Preferably, any such packaging signal(s) aredeleted from all of the helper nucleic acids.

Production of Alphavirus Particles

Alphavirus replicon particles of this invention are produced byintroducing helper constructs and replicon nucleic acids into a helpercell so that the helper and replicon molecules function to producealphavirus replicon particles. In embodiments utilizing RNA helpers, thehelpers can be introduced into the cells in a number of ways. The RNAscan be introduced as RNA or DNA molecules that can be expressed in thehelper cell without integrating into the cell genome. Methods ofintroduction include electroporation, viral vectors (e.g. SV40,adenovirus, nodavirus, astrovirus), and lipid-mediated transfection.Alternatively, they can be expressed from one or more expressioncassettes that have been stably transformed into the cells, therebyestablishing packaging cell lines (see, for example, U.S. Pat. No.6,242,259).

In other embodiments, the helper is a single DNA molecule which encodesall the polypeptides necessary for packaging the viral replicon RNA intoinfective alphavirus replicon particles. The single DNA helper can beintroduced into the packaging cell by any means known to the art,including by electroporation, typically with an increase in voltage ascompared to that required for the uptake of RNA, but a voltage notsufficiently high to destroy the ability of the packaging cells toproduce infectious alphavirus replicon particles. The DNA helper can beintroduced prior to, concomitantly, with, or afterintroduction/expression of the alphavirus RNA vector replicon.Alternatively, the helper function, in this format and under aninducible promoter, can be incorporated into the packaging cell genomeprior to the introduction/expression of the alphavirus RNA vectorreplicon, and then induced with the appropriate stimulus just prior to,concomitant with, or after the introduction of the alphavirus RNA vectorreplicon.

Recombinant DNA molecules that express the alphavirus structuralproteins can also be generated from a single helper that resolves itselfinto two separate molecules in vivo. Thus, the advantage of using asingle helper in terms of ease of manufacturing and efficiency ofproduction is preserved, while the advantages of a bipartite helpersystem are captured in the absence of employing a bipartite expressionsystem. A DNA helper construct can be used, while in a second set an RNAhelper vector is used. Such systems are described in detail in Smith etal. “Alphavirus Replicon Vector Systems”, U.S. Patent Publication2003-0119182A1, incorporated herein by reference.

For the DNA helper constructs, a promoter for directing transcription ofRNA from DNA, i.e. a DNA dependent RNA polymerase, is employed. In thepresent context, a promoter is a sequence of nucleotides recognized by apolymerase and sufficient to cause transcription of an associated(downstream) sequence. In some embodiments of the claimed invention, thepromoter is constitutive (see below). Alternatively, the promoter may beregulated, i.e., not constitutively acting to cause transcription of theassociated sequence. If inducible, there are sequences present whichmediate regulation of expression so that the associated sequence istranscribed only when (i) an inducer molecule is present in the mediumin or on which the cells are cultivated, or (ii) conditions to which thecells are exposed are changed to be inducing conditions. In the presentcontext, a transcription regulatory sequence includes a promotersequence and can further include cis-active sequences for regulatedexpression of an associated sequence in response to environmentalsignals.

In the RNA helper embodiments, the promoter is utilized to synthesizeRNA in an in vitro transcription reaction, and specific promoterssuitable for this use include the SP6, T7, and T3 RNA polymerasepromoters. In the DNA helper embodiments, the promoter functions withina cell to direct transcription of RNA. Potential promoters for in vivotranscription of the construct include eukaryotic promoters such as RNApolymerase II promoters, RNA polymerase III promoters, or viralpromoters such as MMTV and MoSV LTR, SV40 early region, RSV or CMV. Manyother suitable mammalian and viral promoters for the present inventionare available in the art. Alternatively, DNA dependent RNA polymerasepromoters from bacteria or bacteriophage, e.g. SP6, T7, and T3, may beemployed for use in vivo, with the matching RNA polymerase beingprovided to the cell, either via a separate plasmid, RNA vector, orviral vector. In a specific embodiment, the matching RNA polymerase canbe stably transformed into a helper cell line under the control of aninducible promoter.

DNA constructs that function within a cell can function as autonomousplasmids transfected into the cell or they can be stably transformedinto the genome. In these embodiments, the promoter may be aconstitutive promoter, i.e. a promoter which, when introduced into acell and operably linked to a downstream sequence, directs transcriptionof the downstream sequence upon introduction into the cell, without theneed for the addition of inducer molecules or a change to inducingconditions. Alternatively, the promoter may be inducible, so that thecell will only produce the functional messenger RNA encoded by theconstruct when the cell is exposed to the appropriate stimulus(inducer). When using an inducible promoter, the helper constructs areintroduced into the packaging cell concomitantly with, prior to, orafter exposure to the inducer, and expression of the alphavirusstructural proteins occurs when both the constructs and the inducer arepresent. Alternatively, constructs designed to function within a cellcan be introduced into the cell via a viral vector, e.g. adenovirus,poxvirus, adeno-associated virus, SV40, retrovirus, nodavirus,picornavirus, vesicular stomatitis virus, and baculoviruses withmammalian pol II promoters.

Once an RNA transcript (mRNA) encoding the helper or alphavirus RNAreplicon vectors of this invention is present in the helper cell (eithervia in vitro or in vivo approaches, as described above), it iseventually translated to produce the encoded polypeptides or proteins.In certain embodiments, the alphavirus RNA vector replicon istranscribed in vitro from a DNA plasmid and then introduced into thehelper cell by electroporation. In other embodiments, the RNA vectorreplicon of this invention is transcribed in vivo from a DNA vectorplasmid that is transfected into the helper cell (e.g. see U.S. Pat. No.5,814,482), or it is delivered to the helper cell via a virus orvirus-like particle.

In the embodiments of this invention, one or more of the nucleic acidsencoding the alphavirus RNA replicon or helpers is comprised ofsequences derived from the VEETC83 genome, which contains mutations thatcontribute to the attenuated nature of the TC83 vaccine strain, asdescribed hereinabove. In addition, one or more of the nucleic acidsencoding the alphavirus structural proteins, i.e., the capsid, E1glycoprotein and E2 glycoprotein, or the replicon construct, may containone or more additional attenuating mutations.

Methods for Immunizing Subjects

As used herein, “eliciting an immune response” and “immunizing asubject” includes the development, in a subject, of a humoral and/or acellular immune response to a protein and/or polypeptide produced by theparticles and/or compositions of this invention (e.g. an immunogen, anantigen, an immunogenic peptide, and/or one or more epitopes). A“humoral” immune response, as this term is well known in the art, refersto an immune response comprising antibodies, while a “cellular” immuneresponse, as this term is well known in the art, refers to an immuneresponse comprising T-lymphocytes and other white blood cells,especially the immunogen-specific response by HLA-restricted cytolyticT-cells, i.e., “CTLs.” A cellular immune response occurs when theprocessed immunogens, i.e., peptide fragments, are displayed inconjunction with the major histocompatibility complex (MHC) HLAproteins, which are of two general types, class I and class II. Class IHLA-restricted CTLs generally bind 9-mer peptides and present thosepeptides on the cell surface. These peptide fragments in the context ofthe HLA Class I molecule are recognized by specific T-Cell Receptor(TCR) proteins on T-lymphocytes, resulting in the activation of theT-cell. The activation can result in a number of functional outcomesincluding, but not limited to, expansion of the specific T-cell subsetresulting in destruction of the cell bearing the HLA-peptide complexdirectly through cytotoxic or apoptotic events or the activation ofnon-destructive mechanisms, e.g., the production ofinterferon/cytokines. Presentation of immunogens via Class I MHCproteins typically stimulates a CD8+CTL response.

Another aspect of the cellular immune response involves the HLA ClassII-restricted T-cell responses, involving the activation of helperT-cells, which stimulate and focus the activity of nonspecific effectorcells against cells displaying the peptide fragments in association withthe MHC molecules on their surface. At least two types of helper cellsare recognized: T-helper 1 cells (Th1), which secrete the cytokinesinterleukin 2 (IL-2) and interferon-gamma and T-helper 2 cells (Th2),which secrete the cytokines interleukin 4 (IL-4), interleukin 5 (IL-5),interleukin 6 (IL-6) and interleukin 10 (IL-10). Presentation ofimmunogens via Class II MHC proteins typically elicits a CD4+CTLresponse as well as stimulation of B lymphocytes, which leads to anantibody response.

An “immunogenic polypeptide,” “immunogenic peptide,” or “immunogen” asused herein includes any peptide, protein or polypeptide that elicits animmune response in a subject and in certain embodiments, the immunogenicpolypeptide is suitable for providing some degree of protection to asubject against a disease. These terms can be used interchangeably withthe term “antigen.”

In certain embodiments, the immunogen of this invention can comprise,consist essentially of or consist of one or more “epitopes.” An“epitope” is a set of amino acid residues which is involved inrecognition by a particular immunoglobulin. In the context of T cells,an epitope is defined as the amino acid residues necessary forrecognition by T cell receptor proteins and/or MHC receptors. In animmune system setting, in vivo or in vitro, an epitope refers to thecollective features of a molecule, such as primary, secondary and/ortertiary peptide structure, and/or charge, that together form a siterecognized by an immunoglobulin, T cell receptor and/or HLA molecule. Inthe case of a B-cell (antibody) epitope, it is typically a minimum of3-4 amino acids, preferably at least 5, ranging up to approximately 50amino acids. Preferably, the humoral response-inducing epitopes arebetween 5 and 30 amino acids, usually between 12 and 25 amino acids, andmost commonly between 15 and 20 amino acids. In the case of a T-cellepitope, an epitope includes at least about 7-9 amino acids, and for ahelper T-cell epitope, at least about 12-20 amino acids. Typically, sucha T-cell epitope will include between about 7 and 15 amino acids, e.g.,7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids.

The alphavirus particles of this invention are employed to express anucleic acid encoding an immunogenic polypeptide in a subject (e.g., forvaccination) or for immunotherapy (e.g., to treat a subject with canceror tumors). Thus, in the case of vaccines, the present invention therebyprovides methods of eliciting an immune response in a subject,comprising administering to the subject an immunogenic amount of apopulation of alphavirus particles.

An “immunogenic amount” is an amount of the infectious alphavirusparticles which is sufficient to evoke an immune response in the subjectto which the pharmaceutical formulation comprising the alphavirusparticles is administered. An amount of from about 10⁴ to about 10⁹,especially 10⁶ to 10⁸, infectious units, per dose is believed suitable,depending upon the age and species of the subject being treated.Exemplary pharmaceutically acceptable carriers include, but are notlimited to, sterile pyrogen-free water and sterile pyrogen-freephysiological saline solution.

A “subject” of this invention includes, but is not limited to,warm-blooded animals, e.g., humans, non-human primates, horses, cows,cats, dogs, pigs, rats, and mice. Administration of the variouscompositions of this invention (e.g., nucleic acids, particles,populations, pharmaceutical compositions) can be accomplished by any ofseveral different routes. In specific embodiments, the compositions canbe administered intramuscularly, subcutaneously, intraperitoneally,intradermally, intranasally, intracranially, sublingually,intravaginally, intrarectally, orally, or topically. The compositionsherein may be administered via a skin scarification method, ortransdermally via a patch or liquid. The compositions may be deliveredsubdermally in the form of a biodegradable material which releases thecompositions over a period of time.

The compositions of this invention can be used prophylactically toprevent disease or therapeutically to treat disease. Diseases that canbe treated include infectious disease caused by viruses, bacteria, fungior parasites, and cancer. Chronic diseases involving the expression ofaberrant or abnormal proteins or the over-expression of normal proteins,can also be treated, e.g., Alzheimer's, disease multiple sclerosis,stroke, etc.

The compositions of this invention can be optimized and combined withother vaccination regimens to provide the broadest (i.e., all aspects ofthe immune response, including those features described hereinabove)cellular and humoral responses possible. In certain embodiments, thiscan include the use of heterologous prime-boost strategies, in which thecompositions of this invention are used in combination with acomposition comprising a different modality for vaccination, such as oneor more of the following: immunogens derived from a pathogen or tumor,recombinant immunogens, naked nucleic acids, nucleic acids formulatedwith lipid-containing moieties, non-alphavirus vectors (including butnot limited to pox vectors, adenoviral vectors, herpes vectors,vesicular stomatitis virus vectors, paramyxoviral vectors, parvovirusvectors, papovavirus vectors, retroviral vectors), and other alphavirusvectors. The viral vectors can be virus-like particles or nucleic acids.The alphavirus vectors can be replicon-containing particles, DNA-basedreplicon-containing vectors (sometimes referred to as an “ELVIS” system,see, for example, U.S. Pat. No. 5,814,482) or naked RNA vectors. Inspecific embodiments, VRPs can be used as a priming inoculation,followed by one or more boosting inoculations using one of theabove-listed compositions. Alternatively, VRPs can be used in one ormore boosting inoculations following a priming inoculation with one ofthe above-listed compositions.

The compositions of the present invention can also be employed toproduce an immune response against chronic or latent infectious agents,which typically persist because they fail to elicit a strong immuneresponse in the subject. Illustrative latent or chronic infectiousagents include, but are not limited to, hepatitis B, hepatitis C,Epstein-Barr Virus, herpes viruses, human immunodeficiency virus, andhuman papilloma viruses. Alphavirus replicon particles of this inventionencoding peptides and/or proteins from these infectious agents can beadministered to a cell or a subject according to the methods describedherein.

Alternatively, the immunogenic protein or peptide can be any tumor orcancer cell antigen. Preferably, the tumor or cancer antigen isexpressed on the surface of the cancer cell. Exemplary cancer antigensfor specific breast cancers are the HER2 and BRCA1 antigens. Otherillustrative cancer and tumor cell antigens are described in S.A.Rosenberg, (1999) Immunity 10:281) and include, but are not limited to,MART-1/MelanA, gp100, tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3,GAGE-1/2, BAGE, RAGE, NY-ESO-1, CDK-4, β-catenin, MUM-1, Caspase-8,KIAA0205, HPVE&, SART-1, PRAME, p15 and p53 antigens, Wilms' tumorantigen, tyrosinase, carcinoembryonic antigen (CEA), prostate specificantigen (PSA), prostate-specific membrane antigen (PSMA), prostate stemcell antigen (PSCA), human aspartyl (asparaginyl) β-hydroxylase (HAAH),and EphA2 (an epithelial cell tyrosine kinase, see International PatentPublication No. WO 01/12172).

The immunogenic polypeptide or peptide of this invention can also be a“universal” or “artificial” cancer or tumor cell antigen as described ininternational patent publication WO 99/51263, which is incorporatedherein by reference in its entirety for the teachings of such antigens.

In various embodiments, the heterologous nucleic acid of this inventioncan encode an antisense nucleic acid sequence. An “antisense” nucleicacid is a nucleic acid molecule (i.e., DNA or RNA) that is complementary(i.e., able to hybridize in vivo or under stringent in vitro conditions)to all or a portion of a nucleic acid (e.g., a gene, a cDNA and/or mRNA)that encodes or is involved in the expression of nucleic acid thatencodes a polypeptide to be targeted for inhibited or reduced productionby the action of the antisense nucleic acid. Where the antisense nucleicacid is complementary to a portion of the nucleic acid encoding thepolypeptide to be targeted, the antisense nucleic acid should hybridizeclose enough to the 5′ end of the nucleic acid encoding the polypeptidesuch that it inhibits translation of a functional polypeptide.Typically, this means that the antisense nucleic acid should becomplementary to a sequence that is within the 5′ half or third of thenucleic acid to which it hybridizes.

An antisense nucleic acid of this invention can also encode a catalyticRNA (i.e., a ribozyme) that inhibits expression of a target nucleic acidin a cell by hydrolyzing an mRNA encoding the targeted gene product.Additionally, hammerhead RNA can be used as an antisense nucleic acid toprevent intron splicing. An antisense nucleic acid of this invention canbe produced and tested according to protocols routine in the art forantisense technology.

Standard techniques for cloning, DNA isolation, amplification andpurification, for enzymatic reactions involving DNA ligase, DNApolymerase, restriction endonucleases and the like, and variousseparation techniques are those known and commonly employed by thoseskilled in the art. A number of standard techniques are described inSambrook et al. (1989) Molecular Cloning, Second Edition, Cold SpringHarbor Laboratory, Plainview, N.Y.; Maniatis et al. (1982) MolecularCloning, Cold Spring Harbor Laboratory, Plainview, N.Y.; Wu (ed.) (1993)Meth. Enzymol. 218, Part I; Wu (ed.) (1979) Meth. Enzymol. 68; Wu et al.(eds.) (1983) Meth. Enzymol. 100 and 101; Grossman and Moldave (eds.)Meth. Enzymol. 65; Miller (ed.) (1972) Experiments in MolecularGenetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Oldand Primrose (1981) Principles of Gene Manipulation, University ofCalifornia Press, Berkeley; Schleif and Wensink (1982) Practical Methodsin Molecular Biology; Glover (ed.) (1985) DNA Cloning Vol. I and II, IRLPress, Oxford, UK; Hames and Higgins (eds.) (1985) Nucleic AcidHybridization, IRL Press, Oxford, UK; Setlow and Hollaender (1979)Genetic Engineering: Principles and Methods, Vols. 1-4, Plenum Press,New York; and Ausubel et al. (1992) Current Protocols in MolecularBiology, Greene/Wiley, New York, N.Y., and in other sources referencedherein. Abbreviations and nomenclature, where employed, are deemedstandard in the field and commonly used in professional journals such asthose cited herein.

All references cited in the present application are incorporated byreference herein to the extent that there is no inconsistency with thepresent disclosure.

When a Markush group or other grouping is used herein, all individualmembers of the group and all combinations and subcombinations possibleof the group are intended to be individually included in the disclosure.Whenever a range is given in the specification, for example, atemperature range, a time range, or a composition range, allintermediate ranges and subranges, as well as all individual valuesincluded in the ranges given are intended to be included in thedisclosure.

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting of” excludes any element, step, or ingredient notspecified in the claim element. As used herein, “consisting essentiallyof” does not exclude materials or steps that do not materially affectthe basic and novel characteristics of the claim. Any recitation hereinof the term “comprising”, particularly in a description of components ofa composition or in a description of elements of a device in thespecification or claims, can be exchanged with “consisting essentiallyof” or “consisting of”.

One of ordinary skill in the art will appreciate that methods,techniques, procedures, e.g., collection and/or purification techniquesor procedures, starting materials, culture media, and reagents otherthan those specifically exemplified can be employed in the practice ofthe invention without resort to undue experimentation. All art-knownfunctional equivalents, of any such methods, techniques, procedures,starting materials, culture media, and reagents are intended to beincluded in this invention.

Although the description herein contains many specific recitations andexamples, these should not be construed as limiting the scope of theinvention, but as merely providing illustrations of some of theembodiments of the invention. All references cited herein are herebyincorporated by reference to the extent that there is no inconsistencywith the disclosure of this specification. Some references providedherein are incorporated by reference herein to provide detailsconcerning additional starting materials, additional methods ofsynthesis, additional methods of analysis and additional uses of theinvention.

The following examples are provided for illustrative purposes, and arenot intended to limit the scope of the invention as claimed herein. Anyvariations in the exemplified articles which occur to the skilledartisan are intended to fall within the scope of the present invention.

EXAMPLES Example 1 Production of TC-83 Replicons

A replicon plasmid based on the TC-83 strain of VEE was produced from aTC-83 infectious cDNA clone, pVE/IC-92, obtained from the Centers forDisease Control and Prevention. The sequence of this clone was publishedby Kinney et al. (1993) J. Virol. 67:1269. The pVE/IC-92 sequencediffers from the TC-83 virus genomic sequence by the presence of anAla-Val mutation at E1-119 (a cloning artifact introduced by Kinney) andthree silent mutations in nsp1 (at 1613A→G; at 1616C→A; at 1619T→C)purposely introduced to distinguish the clone-derived virus from thegenomic sequence. The present inventors have identified an additionalsilent mutation at E1 position in the pVE/IC-92 clone. By “silent” ismeant that the change in the nucleic acid sequence does not cause achange in the amino acid that is encoded by that nucleic acid sequence.

The TC-83 replicon vector (“pVEK”) was produced by first transferring anexpressible sequence encoding kanamycin resistance (“KN(R)”) into theTC-83 full-length clone to create pVEK/IC-92. A multiple cloning sitewas inserted in place of the TC-83 structural protein genes by digestingan existing VEE replicon (such as the pERK plasmid, see U.S. PatentPublication No. 2002-141975, Example 2), which has the VEE 26S promoterand 3′ UTR (untranslated region), with ApaI and NotI restriction enzymesand ligating that fragment into the same sites of pVEK/IC-92. Theresulting plasmid is replicated in bacteria using the COLE1 origin ofreplication (ORI) and contains the TC-83 5′ and 3′ UTR's, TC 83nonstructural protein (nsP) sequences, a VEE 26S promoter, and amultiple cloning site, all placed downstream of a T7 polymerase promoterfor in vitro RNA transcription.

Alternatively, the structural proteins of the TC-83 clone were replacedwith a chimeric heterologous gene, e.g. either the HIV gag (GAG) gene,the gene encoding the green fluorescent protein (GFP), or an alphavirus(VEE, EEE or WEE) glycoprotein polyprotein sequence.

A second TC-83 based replicon was produced in which the VEE 26S promoterdrives transcription of the heterologous gene, while an internalribosome entry site (IRES) was inserted downstream of the promoter isused to direct translation from the subgenomic RNA (herein referred toas an “IRES replicon” and specifically “VEETC83IRES”). This replicon wasgenerated from pERK-342EnGGAG (herein also referred to as“VEE3000IRES”), which is a wild-type VEE-based replicon that contains a342 bp sequence (SEQ ID NO:1) (an AluI fragment from the digestion ofpcDNA3.1 DNA; Invitrogen, Inc; Carlsbad, Calif.) inserted at the EcoRVrestriction enzyme site of pERK between the subgenomic promoter and EMCVIRES, as an ApaI-SphI fragment into pVEK-IC92. The 342 bp sequence isinserted to insure that the IRES is the control element for translation,and has the following sequence:

CTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTGTATATCCATTTTCGGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAG

The cloning was done in two steps due to the presence of an ApaIrestriction enzyme site in the EMCV IRES.

Replicon plasmids were produced by transforming E. coli with the plasmidand then isolating the DNA plasmid using a Marligen Biosciences(Ijamsville, Md.) High Purity Plasmid Purification System, which uses aproprietary ion exchange resin to yield highly purified plasmid DNA.Alternatively, another DNA purification procedure that results in DNAwhich is free of RNA, protein or endotoxin is acceptable.

Aliquots of the purified replicon plasmid were transcribed in vitro fromNotI linearized plasmid DNA using T7 polymerase. Typically, the T7RiboMAX Express System (Promega, Madison, Wis.), which contains amixture of T7 RNA polymerase, Recombinant RNasin® RNase Inhibitor andyeast inorganic pyrophosphatase that allows for large scale RNAproduction, was used. The resulting RNA was then purified using theRNeasy Midi kit (Qiagen, Valencia, Calif.), which utilizes asilica-gel-based membrane to bind RNA and purify it away fromcontaminating protein. Alternatively, another RNA purification schemewhich results in purified RNA in water that is free of RNases isacceptable.

Example 2 Production of TC-83 Helpers

A. DNA Helper

A TC-83 DNA helper was constructed from pcDNA-VSp, which is described inU.S. Patent Publication No. 2003-0119182, Example 5. pcDNA-VSp is a DNAhelper in which the VEE3014 VEE structural proteins are expresseddirectly from a CMV promoter. The glycoprotein gene sequence containingthe TC-83 mutations was digested from pVE/IC-92 using SpeI and ScaIrestriction enzymes, and ligated into pcDNA-Vsp which has been digestedwith the same enzymes. The introduced mutation at E1-119, which wasnoted but uncorrected by Kinney et al. (1993) J. Virol. supra as anartifact of the cDNA cloning to produce VE/IC-92, was repaired using thequick change site-directed mutagenesis kit (Stratagene, LaJolla, Calif.)and primers TC83E1119F (GCCTTGCGGATCATGCTGAAGCATATAAAGCGC) (SEQ ID NO:2)and TC83E1119R (GCGCTTTATATGCTTCAGCATGATCCGCAAGGC) (SEQ ID NO:3) togenerate pcDNA-TC83r.

E. coli cultures transformed with the DNA helper plasmids were sent toPuresyn, Inc. (Malvern, PA) where they were grown up and the resultingDNA was purified using their POLYFLO® technology (ion-paired, reversephase chromatography resin, Puresyn, Inc., Malvern, PA), resulting in aDNA preparation that was at least 5 mg/ml and free of detectable RNA,ssDNA, linear plasmid or chromosomal DNA.

B. RNA Helpers

The VEE strain TC-83 (“VEETC83”) does not contain any amino acidmutations in the capsid structural protein, so a TC-83 capsid helper canbe constructed from any VEE strain, e.g. as described in Pushko et al.1997 and in U.S. Pat. Nos. 5,792,462; 6,156,558; 5,811,407; and6,008,035. The TC83 glycoprotein helpers were constructed frompcDNA-TC83r, (described above) by digesting with SpeI and NdeI andcloning into a VEE glycoprotein helper RNA (described in the abovereferences and in U.S. Patent Publication No. 2002-0141975, Example 4,incorporated herein by reference) that has been digested with the sameenzymes to remove the 3014 glycoprotein sequence, leaving the 5′ and 3′sequences. In certain embodiments, an additional mutation at E1 81 (fromPhe to Ile) was engineered into the TC-83 glycoprotein helper bysite-directed mutagenesis, referred to herein as “GP-E181I”.

Each helper plasmid was in vitro transcribed from Not I linearizedplasmid DNA using T7 polymerase, exactly as described for the repliconplasmids above.

Example 3 Packaging of TC-83 Replicon with Various Helpers

VEETC83 replicon particles (VRPs) were produced by co-electroporation ofa TC83 replicon RNA (expressing the HIV GAG gene), and one or morehelper nucleic acids (see Table 1) into Vero cells. Followingelectroporation, the cells were seeded into 2 T300 flasks containingOPTIPRO® (serum-free culture medium, Gibco, Carlsbad, CA) and incubatedfor approximately 18 hours. The media was removed from each flask, and10 ml of a 0.5 M salt wash solution in 10 mM sodium phosphate buffer wasadded to each flask and incubated for approximately 5 minutes at roomtemperature before collection and filtration. VRP were titered byincubating serial dilutions of the salt wash and/or the collected mediumon Vero cells in 96 well plates overnight at 37° C. and 5% CO₂. GAG VRPinfected cells were detected using an anti-GAG indirectimmunofluorescence assay on Vero cells fixed with MeOH, and titers weredetermined by counting GAG positive cells at a specific dilution.Similarly, GFP-VRP titers were determined by counting the number of GFPpositive cells at a specific dilution under a UV microscope. The resultsare shown in Table 1.

TABLE 1 Electroporation Inserted RNA Total VRP Yield Helper(s) RepliconConditions encoding: (Salt wash) VEE3014 VEE3000 4 pulses at GAG 3.11e10two RNA helpers (C, 580 V; 25 μF GP) VEE3014 VEE3000 4 pulses at GFP2.13e10 two RNA helpers (C, 580 V; 25 μF GP) VEE3014 VEETC83 4 pulses atGAG  2.94e10 two RNA helpers (C, 580 V; 25 μF. GP) VEE3014 VEETC83 4pulses at GFP 1.41e10 two RNA helpers (C, 580 V; 25 μF. GP) pCDNA-TC83rVEE3000 1 pulse at GAG  1.2e9 250 V; 950 μF pCDNA-TC83r VEETC83 1 pulseat GAG  9.6e8 250 V; 950 μF VEETC83 VEE3000 4 pulses at GAG 5.98e10 twoRNA helpers (C, 580 V; 25 μF. GP) VEETC83 VEETC83 4 pulses at GAG1.15e11 two RNA helpers (C, 580 V; 25 μF. GP) VEETC83 VEE3000IRES** 4pulses at GAG 2.85e10 two RNA helpers (C, 580 V; 25 μF. GP) VEE TC-83VEETC83IRES** 4 pulses at GAG 3.43e10 two RNA helpers (C, 580 V; 25 μF.GP) VEE TC-83 VEETC83IRES** 4 pulses at GAG  1.6e10 two RNA helpers (C,580 V; 25 μF. GP) VEE TC-83 VEETC83IRES** 4 pulses at GAG  2.4e9 two RNAhelpers (C, 580 V; 25 μF. GP-E181I) ** IRES = contains replicon in whichtranslation of heterologous gene is under the control of an IRES

Example 4

A. Packaging of VEETC83 Replicons Expressing Various HeterologousAlphavirus Glycoprotein Genes with TC-83 Structural Proteins

Replicons expressing various heterologous nucleic acids were packagedusing a single TC-83 DNA helper expressing the entire alphavirusstructural polyprotein from the TC-83 strain. In this example, theheterologous genes were glycoprotein cassettes from other alphaviruses.Also included were TC-83 replicons expressing VEE glycoproteins fromeither the TC-83 or 3014 strain. In each of these constructs, theglycoprotein-encoding heterologous nucleic acid comprises theE3-E2-6k-E1 polyprotein from the respective virus. The glycoproteincassettes are identified as follows: “WEE CBA87”, from Western equineencephalitis virus strain Cba 87 and “EEE4002” from Eastern equineencephalitis virus strain Florida 91.

For the WEE cassette, the nucleotide sequence of the WEE virusglycoprotein genes (strain Cba 87) was cloned into the TC-83 replicon,starting from the amino-terminal serine codon of E3 through to thecarboxy terminal arginine codon of E1 (SEQ ID NO:4), as follows:

TCACTAGTTACAGCGCTGTGCGTGCTTTCGAATGTCACATTCCCTTGCGACAAACCACCCGTGTGCTATTCACTGGCGCCAGAACGAACACTCGACGTGCTCGAGGAGAACGTCGACAATCCAAATTACGACACGCTGCTGGAGAACGTCTTGAAATGTCCATCACGCCGGCCCAAACGAAGCATTACCGATGACTTCACGCTGACCAGTCCCTACCTGGGGTTCTGCCCGTATTGCAGACACTCAGCGCCATGTTTTAGCCCAATAAAAATTGAGAACGTGTGGGACGAATCTGATGATGGGTCGATTAGAATCCAGGTCTCGGCACAATTCGGCTACAATCAGGCAGGCACTGCAGACGTCACCAAGTTCCGGTACATGTCTTACGACCACGACCATGACATCAAGGAAGACAGTATGGAGAAATTAGCTATTAGTACATCCGGACCATGCCGTCGTCTTGGCCACAAAGGGTACTTCCTGTTAGCTCAATGTCCTCCAGGTGACAGTGTAACCGTCAGTATCACGAGCGGAGCATCTGAGAATTCATGCACCGTGGAGAAAAAGATCAGGAGGAAGTTTGTCGGTAGAGAGGAGTACTTGTTCCCACCTGTCCATGGAAAGCTGGTAAAGTGCCACGTTTACGATCACTTGAAGGAGACGTCTGCCGGATATATAACTATGCACAGGCCAGGCCCACACGCGTATAAGTCCTACCTGGAGGAAGCGTCAGGCGAAGTGTACATTAAACCACCTTCTGGCAAGAACGTCACCTACGAATGTAAGTGTGGTGACTACAGCACAGGTATTGTGAGCACGCGAACGAAGATGAACGGCTGCACTAAAGCAAAACAATGCATTGCCTACAAGCGCGACCAAACGAAATGGGTCTTCAACTCGCCGGATCTTATTAGGCACACAGACCACTCAGTGCAAGGTAAACTGCACATTCCATTCCGCTTGACACCGACAGTCTGCCCGGTTCCGTTAGCTCACACGCCTACAGTCACGAAGTGGTTCAAAGGCATCACCCTCCACCTGACTGCAACGCGACCAACATTGCTGACAACGAGAAAATTGGGGCTGCGAGCAGACGCAACAGCAGAATGGATTACGGGGACTACATCCAGGAATTTTTCTGTGGGGCGAGAAGGGCTGGAGTACGTATGGGGCAACCATGAACCAGTCAGAGTCTGGGCCCAGGAGTCGGCACCAGGCGACCCGCATGGATGGCCGCATGAGATCATCATCCATTATTATCATCGGCATCCAGTCTACACTGTCATTGTGCTGTGCGGTGTCGCTCTGGCTATCCTGGTAGGCACTGCATCGTCAGCAGCTTGTATCGCCAAAGCAAGAAGAGACTGCCTGACGCCATACGCGCTTGCACCGAACGCAACGGTACCCACAGCATTAGCAGTTTTGTGCTGTATTCGGCCAACCAACGCTGAAACATTTGGAGAAACTTTGAACCATCTGTGGTTTAACAACCAACCGTTTCTCTGGGCACAGTTGTGCATCCCTCTGGCAGCGCTTATTATTCTGTTCCGCTGCTTTTCATGCTGCATGCCTTTTTTATTGGTTGCAGGCGTCTGCCTGGGGAAGGTAGACGCCTTCGAACATGCGACCACTGTGCCAAATGTTCCGGGGATCCCGTATAAGGCGTTGGTCGAACGTGCAGGTTACGCGCCACTTAATCTGGAGATTACGGTCGTCTCATCGGAATTAACACCCTCAACTAACAAGGAGTACGTGACCTGCAAATTTCACACAGTCGTTCCTTCACCACAAGTTAAATGCTGCGGGTCCCTCGAGTGTAAGGCATCCTCAAAAGCGGATTACACATGCCGCGTTTTTGGCGGTGTGTACCCTTTCATGTGGGGAGGCGCACAGTGCTTCTGTGACAGTGAGAACACACAACTGAGTGAGGCATACGTCGAGTTCGCTCCAGACTGCACTATAGATCATGCAGTCGCACTAAAAGTTCACACAGCTGCTCTGAAAGTCGGCCTGCGTATAGTATACGGCAATACCACAGCGCGCCTGGATACATTCGTCAACGGCGTCACACCAGGTTCCTCACGGGACCTGAAGGTCATAGCAGGGCCGATATCAGCAGCTTTTTCACCCTTTGACCATAAGGTCGTCATTAGAAAGGGGCTTGTTTACAACTACGACTTCCCTGAGTATGGAGCTATGAACCCAGGAGCGTTCGGCGATATTCAAGCATCCTCTCTTGATGCCACAGACATAGTAGCCCGCACCGACATACGGCTGCTGAAGCCTTCTGTCAAGAACATCCACGTCCCCTACACCCAAGCAGTATCAGGGTATGAAATGTGGAAGAACAACTCAGGACGACCCCTGCAAGAAACAGCACCATTCGGATGTAAAATTGAAGTGGAGCCTCTGCGAGCGACTAACTGTGCTTATGGGCACATCCCTATCTCGATTGACATCCCTGATGCAGCTTTTGTGAGATCATCTGAATCACCAACAATTTTAGAAGTCAGCTGCACAGTAGCAGACTGCATTTATTCTGCAGACTTTGGTGGTTCGCTAACACTACAGTACAAAGCTAACAGAGAGGGACATTGTCCAGTTCACTCCCACTCCACTACAGCTGTTTTGAAGGAAGCGACCACACATGTGACTGCCACAGGCAGCATAACACTACATTTTAGCACATCGAGCCCACAAGCAAATTTCATAGTTTCGCTATGCGGCAAGAAGACCACCTGCAATGCTGAATGTAAACCACCGGCCGACCACATAATTGGAGAACCACATAAGGTCGACCAAGAATTCCAGGCGGCAGTTTCCAAAACATCTTGGAACTGGCTGCTTGCACTGTTTGGGGGAGCATCATCCCTCATTGTTGTAGGACTTATAGTGTTGGTCTGCAGCTCTATGCTTATAAACACACGTAGA

For the EEE cassette, the nucleotide sequence of the EEE virusglycoprotein genes (strain Florida 91) was cloned into the TC-83replicon, starting from the amino-terminal serine codon of E3 through tothe carboxy-terminal histidine codon of E1 (SEQ ID NO:5), as follows:

TCGCTCGCCACTGTTATGTGCGTCCTGGCCAATATCACGTTTCCATGTGATCAACCACCCTGCATGCCATGCTGTTATGAAAAGAATCCACACGAAACACTCACCATGCTGGAACAGAATTACGACAGCCGAGCCTATGATCAGCTGCTCGATGCCGCTGTGAAATGTAATGCTAGGAGAACCAGGAGAGATTTGGACACTCATTTCACCCAGTATAAGTTGGCACGCCCGTATATTGCTGATTGCCCTAACTGTGGGCATAGTCGGTGCGACAGCCCTATAGCTATAGAAGAAGTCAGAGGGGATGCGCATGCAGGAGTCATCCGCATCCAGACATCAGCTATGTTCGGTCTGAAGACGGATGGAGTCGATTTGGCCTACATGAGTTTCATGAACGGCAAAACGCAGAAATCAATAAAGATCGACAACCTGCATGTGCGCACCTCAGCCCCTTGTTCCCTCGTGTCGCACCACGGCTATTACATCTTGGCTCAATGCCCACCAGGGGACACGGTTACAGTTGGGTTTCACGACGGGCCTAACCGCCATACGTGCACAGTTGCCCATAAGGTAGAATTCAGGCCAGTGGGTAGAGAGAAATACCGTCACCCACCTGAACATGGAGTTGAACTACCGTGTAACCGTTACACTCACAAGCGTGCAGACCAAGGACACTATGTTGAGATGCATCAACCAGGGCTAGTTGCCGACCACTCTCTCCTTAGCATCCACAGTGCCAAGGTGAAAATTACGGTACCGAGCGGCGCCCAAGTGAAATACTACTGCAAGTGTCCAGATGTACGAGAGGGAATTACCAGCAGCGACCATACAACCACCTGCACGGATGTCAAACAATGCAGGGCTTACCTGATTGACAACAAGAAATGGGTGTACAACTCTGGAAGACTGCCTCGAGGAGAGGGCGACACTTTTAAAGGAAAACTTCATGTGCCCTTTGTGCCTGTTAAGGCCAAGTGCATCGCCACGCTGGCACCGGAGCCTCTAGTTGAGCACAAACACCGCACCCTGATTTTACACCTGCACCCGGACCATCCGACCTTGCTGACGACCAGGTCACTTGGAAGTGATGCAAATCCAACTCGACAATGGATTGAGCGACCAACAACTGTCAATTTCACAGTCACCGGAGAAGGGTTGGAGTATACCTGGGGAAACCATCCACCAAAAAGAGTATGGGCTCAAGAGTCAGGAGAAGGGAACCCACATGGATGGCCGCACGAAGTGGTAGTCTATTACTACAACAGATACCCGTTAACCACAATTATCGGGTTATGCACCTGTGTGGCTATCATCATGGTCTCTTGTGTCACATCCGTGTGGCTCCTTTGCAGGACTCGCAATCTTTGCATAACCCCGTATAAACTAGCCCCGAACGCTCAAGTCCCAATACTCCTGGCGTTACTTTGCTGCATTAAGCCGACGAGGGCAGACGACACCTTGCAAGTGCTGAATTATCTGTGGAACAACAATCAAAACTTTTTCTGGATGCAGACGCTTATCCCACTTGCAGCGCTTATCGTATGCATGCGCATGCTGCGCTGCTTATTTTGCTGTGGGCCGGCTTTTTTACTTGTCTGCGGCGCCTTGGGCGCCGCAGCGTACGAACACACAGCAGTGATGCCGAACAAGGTGGGGATCCCGTATAAAGCTTTAGTCGAACGCCCAGGTTATGCACCCGTTCATCTACAGATACAGCTGGTTAATACCAGGATAATTCCATCAACTAACCTGGAGTACATCACCTGCAAGTACAAGACAAAAGTGCCGTCTCCAGTAGTGAAATGCTGCGGTGCCACTCAATGTACCTCCAAACCCCATCCTGACTATCAGTGTCAGGTGTTTACAGGTGTTTACCCATTCATGTGGGGAGGAGCCTACTGCTTCTGCGACACCGAAAACACCCAGATGAGCGAGGCGTATGTAGAGCGCTCGGAAGAGTGCTCTATCGACCACGCAAAAGCTTATAAAGTACACACAGGCACTGTTCAGGCAATGGTGAACATAACTTATGGGAGCGTCAGCTGGAGATCTGCAGATGTCTACGTCAATGGTGAAACTCCCGCGAAAATAGGAGATGCCAAACTCATCATAGGTCCACTGTCATCTGCGTGGTCCCCATTCGATAACAAGGTGGTGGTTTATGGGCATGAAGTGTATAATTACGACTTTCCTGAGTACGGCACCGGCAAAGCAGGCTCTTTTGGAGACCTGCAATCACGCACATCAACCAGCAACGATCTGTACGCAAACACCAACTTGAAGCTACAACGACCCCAGGCTGGTATCGTGCACACACCTTTCACCCAGGCGCCCTCTGGCTTCGAACGATGGAAAAGGGACAAAGGGGCACCGTTGAACGACGTAGCCCCGTTTGGCTGTTCGATTGCCCTGGAGCCGCTCCGTGCAGAAAATTGTGCAGTGGGAAGCATCCCTATATCTATAGATATACCCGATGCGGCTTTCACTAGAATATCTGAAACACCGACAGTCTCAGACCTGGAATGCAAAATTACGGAGTGTACTTATGCCTCCGATTTCGGTGGTATAGCCACCGTTGCCTACAAATCCAGTAAAGCAGGAAACTGTCCAATTCATTCTCCATCAGGTGTTGCAGTTATTAAAGAGAATGACGTCACCCTTGCTGAGAGCGGATCATTTACATTCCACTTCTCCACTGCAAACATCCATCCTGCTTTTAAGCTGCAGGTCTGCACCAGTGCAGTTACCTGCAAAGGAGATTGCAAGCCACCGAAAGATCATATCGTCGATTATCCAGCACAACATACCGAATCCTTTACGTCGGCGATATCCGCCACCGCGTGGTCGTGGCTAAAAGTGCTGGTAGGAGGAACATCAGCATTTATTGTTCTGGGGCTTATTGCTACAGCAGTGGTTGCCCTAGTTCTGTTCTTCCATAGACAT

TC-83-VEE replicon particles (VRPs) were produced by co-electroporationof replicon RNA (expressing the indicated alphavirus glycoproteinpolyprotein), and the single DNA helper encoding the TC-83 structuralproteins into 10⁸ Vero cells. Following electroporation, the cells wereseeded into 2 T300 flasks containing OPTIPRO® SFM (serum-free culturemedium, Gibco, Carlsbad, CA) and incubated for approximately 18 hours.The media was then removed from the flask, and 10 mls of a 0.5 M saltwash solution in 10 mM sodium phosphate buffer was added to each flaskand incubated for approximately 5 minutes at room temperature beforecollection and filtration. VRP were titered by incubating serialdilutions of the collected VRP on Vero cells in 96 well plates overnightat 37° C. and 5% CO₂. Alphavirus glycoprotein-expressing VRP infectedcells were detected using an anti-WEE, anti-VEE, or anti-EEE indirectimmunofluorescence assay on Vero cells fixed with 1:1 Acetone:MeOH, andtiters were determined by counting antigen-positive cells at a specificdilution. The results are shown in Table 2.

TABLE 2 TC-83 Replicon (“pVEK”) expressing: Total VRP Yield WECBA87 3.4× 10⁹ EE4002 4.3 × 10⁸ VEE-3014 2.0 × 10⁹ VEE-TC83 3.8 × 10⁹

B. Packaging of VEETC83 and Wild-type VEE Replicons Expressing a Genefrom SARS with VEETC83 or VEE3014 Structural Proteins

The S2 glycoprotein gene from the Severe Acute Respiratory Syndromevirus (“SARS-S2”) was PCR amplified from a SARS coronavirus capsid clone(Urbani strain of SARS coronavirus; Accession # AY278741; obtained fromthe United States Centers for Disease Control and Prevention, Atlanta,Ga.) and inserted into a pERK replicon (described in Example 1 above) asa BamHI restriction fragment immediately downstream of the enterovirus71 (EV71) IRES. This replicon, capable of expressing the SARS-S2glycoprotein gene, was packaged into VRPs using either 3014 or TC83structural proteins. The structural proteins were expressed either fromtwo separate RNA helpers or from a single DNA helper. For the split RNAhelper approach, 30 μg replicon RNA was combined with 30 μg each of theVEE capsid RNA helper and the VEE glycoprotein helper (either fromVEE3014 or VEETC83, see Example 2B above) and co-electroporated into1.2×10⁸ Vero cells. In this experiment, electroporation was carried outin 0.4 cm gap cuvettes using four pulses, each at 580V and 25 μF. Forpackaging with a single DNA helper (encoding the entire sequence ofeither the VEETC83 structural polyprotein or the VEE3014 structuralpolyprotein), 30 μg replicon RNA was combined with 150 μg of the DNAhelper and co-electroporated into 1.2×10⁸ Vero cells in a 0.4 cm gapcuvette, using a single pulse at 250V and 950 μF. VRPs were produced,harvested and tittered as described in Example 3A, and the yields on aper cell basis are reported in Table 3. The yield per cell of VRPs usingTC-83 glycoprotein helpers (as described earlier, the capsid sequence isthe same in both VEETC83 and VEE3014), whether in the RNA or DNA helperformat, was nearly 4 times greater than the yield recovered with 3014glycoprotein helpers.

TABLE 3 SARS-S2 VRP yields from cells electroporated with RNA vs. DNAhelpers expressing the VEE3014 or VEETC83 glycoprotein genes. EP# Helper#1 Helper #2 (amt used) IU/Cell 1 Capsid RNA VEE3014 GP RNA 1000 2Capsid RNA VEETC83 GP RNA 4000 3 pCDNA-VSp NA 51 4 pCDNA-VSp NA 44 5pCDNA-VSp NA 77 6 pCDNA-TC83r NA 260 7 pCDNA-TC83r NA 230 8 pCDNA-TC83rNA 290

C. Enhanced Yield of VRPs Using a VEETC83 DNA Helper

The experiment in 3B. indicated that the VEETC83 DNA helper wasassociated with higher yields of VRPs (compare EP#3-5 with EP#6-8). Thiswas confirmed in a second set of experiments, in which replicon RNAsexpressing either the GAG gene or GFP were packaged with eitherpcDNA-TC83r or the pcDNA-VSp helper (see Table 4). These studies alsoconfirmed that the solution in which the DNA helper was resuspendedprior to co-electroporation (e.g. water (H₂O), phosphate buffered saline(PBS) or Tris EDTA (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) does notsignificantly affect yield, even if the DNA is stored for many months(e.g. 1, 3, 4, 5 or 6 months) at −20° C.

TABLE 4 VRP yields from VEETC83 DNA helper vs. VEE3014 DNA helper EP#Replicon (amount used) Helper (amt used) IU/Cell 1 VEE3000/GFP (30 μg)pCDNA-VSp (H₂O) (150 μg) 43 2 VEE3000/GFP (30 μg) pCDNA-VSp (PBS) (150μg) 23 3 VEE3000/GFP (30 μg) pCDNA-VSp (TE) (150 μg) 51 4 VEE3000/GFP(30 μg) pCDNA-TC83r (H₂O) (150 μg) 130 5 VEE3000/GFP (30 μg) pCDNA-TC83r(PBS) (150 μg) 100 6 VEE3000/GFP (30 μg) pCDNA-TC83r (TE) (150 μg) 98 7VEE3000(nt3A)IRES/Gag (30 μg) pCDNA-VSp (H₂O) (150 μg) 90 8VEE3000(nt3A)IRES/Gag (30 μg) pCDNA-VSp (PBS) (150 μg) 60 9VEE3000(nt3A)IRES/Gag (30 μg) pCDNA-VSp (TE) (150 μg) 110 10VEE3000(nt3A)IRES/Gag (30 μg) pCDNA-TC83r (H₂O) (150 μg) 200 11VEE3000(nt3A)IRES/Gag (30 μg) pCDNA-TC83r (PBS) (150 μg) 250 12VEE3000(nt3A)IRES/Gag (30 μg) pCDNA-TC83r (TE) (150 μg) 300 13VEE3000(nt3A)IRES/Gag (30 μg) pCDNA-VSp (H₂O) (150 μg) 74 14VEE3000(nt3A)IRES/Gag (30 μg) pCDNA-VSp (PBS) (150 μg) 73 15VEE3000(nt3A)IRES/Gag (30 μg) pCDNA-VSp (TE) (150 μg) 68 16VEE3000(nt3A)IRES/Gag (30 μg) pCDNA-TC83r (H₂O) (150 μg) 150 17VEE3000(nt3A)IRES/Gag (30 μg) pCDNA-TC83r (PBS) (150 μg) 190 18VEE3000(nt3A)IRES/Gag (30 μg) pCDNA-TC83r (TE) (150 μg) 200

Example 5 Heparin Affinity Chromatography of TC-83 VRPs

TC-83 VRPs, collected from Vero cells via a 1 M salt wash of the cells,were diluted with 16 mM sodium phosphate (SP) pH 7.4 to a sodiumchloride concentration of 0.12 M or less. The solution was then loadedonto a column containing Heparin sepharose fast flow resin (Amersham) ata linear velocity of 5 ml/min. The TC-83 VRPs were eluted with a lineargradient of increasing sodium chloride concentration (approximately 120mM to 1 M). The TC-83 VRPs elute at a sodium chloride concentration ofapproximately 3 M at a pH of 7.4. FIG. 1 shows the elution profile ofTC-83 VRPs purified by this method. The sharp UV peak (between fractions14 and 23) corresponds to VRP elution. The fractions containing greaterthen 1e9 VRP (fractions 18-23) were collected from the column andformulated by direct dilution in 1% human serum albumin and 5% sucrose.Prior to formulation, 1e8 infectious units (IU) of purified bulk VRPwere fractionated by SDS-PAGE and analyzed by silver stain or westernblot using capsid or glycoprotein specific antibodies to assess purity(FIG. 2). In the silver stained gel, only bands corresponding in size tothe capsid and glycoproteins are evident, indicating a surprising levelof purity.

Example 6 Immunogenicity of TC-83 VRPs

The immunogenicity of TC-83 VRPs has been studied in laboratory animals.

BALB/c mice, five animals per group, were immunized with the indicatedVRP particles by subcutaneous inoculation in the footpad at theindicated dose. The animals were immunized three times (at 3-weekintervals). Humoral responses were measured by GAG ELISA 7-days afterthe first and second booster inoculations, and cellular responses weremeasured by interferon-gamma ELISPOT 7-days after the second boosterinoculation. The ELISA and ELISPOT data presented in Table 6 are thegeometric and arithmetic means, respectively, calculated from the fivemice from each group. The response to the VEE vector was assessed by aVEE neutralization assay (see below).

TABLE 6 Immunogenicity in Mice, Experiment 1. Immunogenicity ELISPOT*ELISA (GMT) (SFCs/1 e6 after after (lymphocytes) GAG VRP** Dose 1^(st)boost 2^(nd) boost after 2^(nd)boost VEE3000* 1 E3 6756 12177 1330VEE3000* 1 E4 13512 27024 1203 VEE3000* 1 E6 40960 48710 1217 VEE3014* 1E4 46 92 212 VEE3014* 1 E6 6756 17222 793 VEETC83 1 E4 243 844 284VEETC83 1 E5 1940 10240 542 VEETC83 1 E6 2941 15521 1050 VEETC83IRES 1E6 1940 13512 449 VEETC83(E181I)IRES 1 E6 8914 23525 607 *The repliconsin the 3000 and 3014 VRPs contain a mutation to A at nucleotide 3 of thereplicon **VEE3000 and VEE3014 VRPs are packaged with wild-type(VEE3000) and VEE3014 structural proteins respectively; VEETC83 VRPscontain a TC83-derived replicon RNA that is packaged with TC83structural proteins

To demonstrate that each mouse of each TC-83 treatment group respondedwith both humoral and cellular immune responses, the ranges for the fiveresponses recorded for each treatment group are presented in thefollowing Table 7:

TABLE 7 Humoral and Cellular Immune Responses (Experiment 1, Mice)ELISPOT ELISA range range Post- Post- Post- GAG VRP Replicon Dose boost1 boost 2 boost 2 VEETC83 1 e4  80-640  640-2560 173-473 VEETC83 1 e5 640-2560  5120-20480 252-838 VEETC83 1 e6 2560-5120 10240-20480 724-1577 VEETC83IRES 1 e6 1280-5120  5120-40960 202-723VEETC83(E181I)IRES 1 e6  5120-10240 10240-40960 269-880 VEE3014IRES 1 e62560-5120 10240-20480 609-933

TABLE 8 Humoral and Cellular Responses (Experiment 2, mice)Immunogenicity ELISPOT* ELISA (SFCs/1 e6 (GMT) (lymphocytes) GAG VRP VEEafter 1^(st) after 2^(nd) after 2^(nd) strain Route Dose boost boostboost 3014IRES*** footpad 1e6 10240 37924 910 3014IRES footpad 1e7 1351270225 970 3014IRES footpad 5e7 20480 67202 1529 TC-83IRES footpad 1e63378 11763 654 TC-83IRES footpad 1e7 8127 27869 787 TC-83IRES footpad5e7 9554 37924 951 TC-83(E181- footpad 1e6 4389 12902 713 I)IRESTC-83(E181- footpad 1e7 6640 30433 1121 I)IRES TC-83(E181- footpad 5e710240 36491 872 I)IRES TC-83IRES intra- 1e6 300 3335 672 muscularTC-83IRES intra- 1e7 2903 6451 1877 muscular TC-83IRES intra- 5e7 440023525 1297 muscular TC-83(E181- intra- 1e6 304 1781 800 I)IRES muscularTC-83(E181- intra- 1e7 5881 40960 1666 I)IRES muscular TC-83(E181-intra- 5e7 20480 54047 1023 I)IRES muscular *ELISPOT numbers areaverages **10 animals/group for the footpad injections and 5/group inintramuscular injections ***no nt3 mutation

TABLE 9 Anti-Vector Response (for animals in Table 8) Anti-VectorResponse to GFP VRP (GMT) GAG VRP VEE Dose/ after after strain Route1^(st) boost 2^(nd) boost 3014IRES* 1 e6/fp 320 830 3014IRES 1 e7/fp28963 34443 3014IRES 5 e7/fp 40960 40960 TC-83IRES 1 e6/fp 1 1 TC-83IRES1 e7/fp 8 274 TC-83IRES 5 e7/fp 1576 2195 TC-83(E1 81-I)IRES 1 e6/fp 1 1TC-83(E1 81-I)IRES 1 e7/fp 7 640 TC-83(E1 81-I)IRES I 5 e7/fp 1114 1280TC-83IRES 1 e6/im 1 1 TC-83IRES 1 e7/im 2 9 TC-83IRES 5 e7/im 29 160TC-83(E1 81-I)IRES 1 e6/im 1 1 TC-83(E1 81-I)IRES 1 e7/im 3 40 TC-83(E181-I)IRES 5 e7/im 1689 2560

Primate studies were also carried out. The immunogenicity of a TC-83replicon vaccine containing the same HIV Glade C gag gene, was conductedin cynomolgus macaques at the Southern Research Institute, Frederick,Md. The construct used in this study was a TC-83 IRES replicon asdescribed above, containing the EMCV IRES, and a 342 nucleotide spacersequence (see Example 1). Each vaccine was administered to six animalsby subcutaneous and intramuscular injection (three animals/route).Animals received three inoculations of 1×10⁸ vaccine particles at 0, 1and 6 months. Humoral immune responses to gag were analyzed 2 weeksafter each booster inoculation, as well as 20 weeks after the firstbooster, i.e. prior to the second booster. Anti-vector responses werealso measured (see Example 6C). Additional safety data were obtainedthrough clinical chemistries and hematology (hemoglobin, WC, plateletcount) which was conducted two weeks after each inoculation.

TABLE 10 Immunization in Primates: ELISA Responses for CynomolgusMacaques (individual animals): Route of ELISA (titer) AdministrationAnimal # 2 wk PB1 20 wk PB1 2 wk PB2 s.c. 1 80 10 40 s.c 2 10 80 40 s.c.3 160 10 320 i.m. 4 10 10 320 i.m. 5 640 80 5120 i.m. 6 640 10 5120

TABLE 11 ELISA GMT (Geometric Mean Titer, Cynomolgus Macaques): Route of2 wk PB1 20 wk PB1 2 wk PB2 Administration GMT GMT GMT Subcutaneous 50.420 80.0 Intramuscular 160 20 2031.9

Cellular immunity was also measured in the primate model. Anti-Gag Tcell responses in cynomolgus macaques vaccinated with various VRPs (intowhich the HIV gag coding sequence was expressed) were measured usinginterferon-gamma ELISPOT assays using pools of overlapping 9mer or 15merpeptides from the HIV Gag protein. Data are presented in Table 12 as thenumber of positively responding animals/total animals receiving thatvaccination protocol. Positively responding animals were defined asthose whose responses were greater than 10 spots after backgroundsubtraction of the responses to irrelevant peptide pools.

TABLE 12 Anti-Gag T cell responses in Cynomolgus Macaques GAG VRP 2 wk 4wk 2 wk 4 wk 8 wk 24 wk 2 wk 4 wk Replicon* Route PP PP PB1 PB1 PB1 PB1PB2 PB2 VEE3014 s.c. 1/3 0/3 1/3 1/3 1/3 0/3 1/3 0/3 IRES i.m. 0/3 0/30/3 0/3 0/3 0/3 1/3 0/3 VEETC83 s.c. 1/3 0/3 2/3 3/3 3/3 0/3 2/3 1/3IRES i.m. 1/3 0/3 2/3 1/3 1/3 1/3 2/3 1/3 PP = post-prime, PB1 =post-boost 1, PB2 = post-boost 2 *Replicon packaged with homologoushelper(s)

Macaque T cell responses were also analyzed utilizing intracellularcytokine staining (ICS) analysis, in which cells were purified 6 weekspost-boost 2 and analyzed for IL-2 and IL-4 production in response toGag overlapping 15mer peptides by ICS. The results are presented inTable 13, where positively responding animals were defined as thosewhose responses were at least 2 times the response to irrelevant peptidepools.

TABLE 13 ICS Analysis of Vaccinated Macaques. GAG VRP CD4 CD8 RepliconRoute IL-4 IL-2 IL-4 IL-2 VEE3014 s.c. 1/3 1/3 2/3 1/3 IRES i.m. 0/3 0/30/3 0/3 VEETC83 s.c. 3/3 2/3 1/3 1/3 IRES i.m. 2/3 0/3 0/3 0/3

The cumulative T cell responses in macaques are summarized in Table 14.

TABLE 14 Summary: Gag specific T cell responses In Macaques GAG VRPReplicon Route Positive animals/group VEE3014 IRES s.c. 3/3 i.m. 1/3VEETC83 IRES s.c. 3/3 i.m. 2/3

Humoral immunity was determined using a Gag-specific ELISA(enzyme-linked immunosorbent assay). Purified recombinanthistidine-tagged (his)-p55 from HIV-1 subtype C isolate DU-422 (AIDSRes. Hum. Retroviruses. 2003 February; 19(2):133-44) was used as thecoating antigen. Briefly, BHK cells were transfected with VEE repliconRNA expressing his-p55, and Triton-X 100 lysates were prepared. Proteinwas purified by metal ion affinity (nickel) chromatography using acommercially available resin and according to the supplier'sinstruction.

Murine sera, 7 days post boost, were evaluated for the presence ofGag-specific antibodies by a standard indirect ELISA. For detection ofGag-specific total Ig, a secondary polyclonal antibody that detects IgM,IgG and IgA was used for end point titer determination. Briefly, 96-wellMaxisorp ELISA plates (Nunc, Naperville, Ill.) were coated with 50 μl of0.05 M sodium carbonate buffer, pH 9.6 (Sigma Chemical Co., St. Louis,Mo.) containing 40-80 ng his-p55 per well. Plates were covered withadhesive plastic and incubated overnight at 4° C. The next day, unboundantigen was discarded, and plates were incubated for 1 hour with 200 μlblocking buffer (PBS containing 3% w/v BSA) at room temperature. Wellswere washed 6 times with PBS and 50 μl of test serum, diluted seriallytwo-fold in buffer (PBS with 1% w/v BSA and 0.05% v/v Tween 20), wasadded to antigen-coated wells. Mouse anti-p24 monoclonal antibody(Zeptometrix, Buffalo, N.Y.) was included in every assay as a positivecontrol. Negative controls in each assay included blanks (wells with allreagents and treatments except serum) and pre-bleed sera. Plates wereincubated for one hour at room temperature, and then rinsed 6 times withPBS. 50 μl/well of alkaline phosphatase (AP)-conjugated goat anti-mousepoly-isotype secondary antibody (Sigma) diluted to a predeterminedconcentration in diluent buffer was added to each well and incubated for1 hour at room temperature. Wells were rinsed 6 times with PBS beforeaddition of 100 μL p-nitrophenyl phosphate (pNPP) substrate (Sigma). Theserum antibody ELISA titer was defined as the inverse of the greatestserum dilution giving an optical density at 405 nm greater than or equalto 0.2 above the background (blank wells).

GAG antigen-specific Interferon-gamma (IFN-γ) secreting cells weredetected using an IFN-γELISPOT Assay. Single-cell suspensions of spleniclymphocytes from TC-83 VRP-GAG-immunized BALB/c mice were prepared byphysical disruption of the splenic capsule in R-10 medium (RPMI medium1640 supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 0.1mM MEM non-essential amino acids solution, 0.01 M HEPES, 2 mM glutamineand 10% heat inactivated fetal calf serum). Lymphocytes were isolated byLympholyte M density gradient centrifugation (Accurate Scientific,Westbury, N.Y.), washed twice and resuspended in fresh R-10 medium.Total, unseparated splenic lymphocyte populations were tested.

A mouse IFN γELISPOT kit (Monoclonal Antibody Technology, Nacka, Sweden)was used to perform the assay. Viable cells were seeded into individualELISPOT wells in a Multiscreen Immobilon-P ELISPOT plate (ELISPOTcertified 96-well filtration plate, Millipore, Bedford, Mass.) that hadbeen pre-coated with an anti-IFN-γ monoclonal antibody, and incubatedfor 16-20 hours. Cells were removed by multiple washes with buffer andthe wells were incubated with a biotinylated anti-IFN-γ monoclonalantibody, followed by washing and incubation withAvidin-Peroxidase-Complex (Vectastain ABC Peroxidase Kit, VectorLaboratories, Burlingame, Calif.). Following incubation, the wells werewashed and incubated for 4 minutes at room temperature with substrate(Avidin-Peroxidase Complex tablets, Sigma) to facilitate formation ofspots, which represent the positions of the individual IFN-γ-secretingcells during culture. Plates were enumerated by automated analysis witha Zeiss KS ELISPOT system.

To enumerate Gag-specific IFN-γ secreting cells in lymphocytes from miceimmunized with various VRP constructs expressing gag, lymphocytes werestimulated with the immunodominant CD8H-2K^(d)-restricted HIV-Gagpeptide, or an irrelevant CD8H-2K^(d)-restricted Influenza-HA peptidefor 16-20 hours (5% CO₂ at 37° C.). The peptides were tested at 10 μg/mland the nef control was tested at 20 μg/ml. Cells minus peptide serve asa background control. As a positive control, cells were stimulated with4 μg/mL concanavalin A for a similar time period. Peptides weresynthesized and purified to >90% at New England Peptide.

C. VEE Neutralization Assay

Neutralizing antibody activity against Venezuelan equine encephalitis(VEE) virus was measured in serum samples of immunized animals (mice orcynomolgus monkeys) using VEE replicon particles (VRP). This test isdesigned to assess the prevention of productive VRP infection ofVRP-susceptible cells by neutralizing antibodies that are present in theserum. In this assay, a defined quantity of propagation-defective VRPexpressing green fluorescent protein (GFP) is mixed with serialdilutions of the animal's serum, incubated, and inoculated onto cellmonolayers. Following another period of incubation, the cell monolayersare examined for GFP-positive cells under UV light. The infectivity ofGFP-expressing VRP (“GFP-VRP”) is prevented, or “neutralized”, by VEEvirus specific neutralizing antibodies in the serum.

The assay is performed as follows: Day 1: Serum from immunized animals(mice or cynomolgus monkeys) is heat inactivated at 56° C. for 30minutes, and then serially diluted in media (MEM with Earle's Salts andL-glutamine, Invitrogen 11095072, supplemented with 0.1 mM Non-EssentialAmino Acids, 100 U/ml penicillin and 100 μg/ml streptomycin). Thesedilutions are mixed with a defined quantity (between 5×10³ and 1.5×10⁴)of GFP-VRP and incubated overnight at 4° C. Day 2: 50 μl of theserum:GFP-VRP mixture is added to a 96-well plate of confluent Verocells and incubated at 37° C. for one hour. The serum:GFP-VRP mixture isremoved and replaced with 100 μl of fresh media and incubated overnightat 37° C. Day 3: the number of GFP-positive cells are quantified underUV light. The 80% neutralization level is determined for each sample andis defined as the greatest serum dilution giving a mean GFP-positivecells (GPC) per grid that is less than or equal to 20% of the number ofGPCs per grid in control wells infected with GFP-VRP alone or withGFP-VRP pre-incubated with negative control sera (i.e. pre-immunizationsera).

TABLE 15 Anti-VEE responses in Mice immunized with GAG-VRP Anti-VectorResponse (GMT) after after GAG VRP VEE strain Dose 1^(st) boost 2^(nd)boost TC-83 1 e4  1* 1 TC-83 1 e5 1 1 TC-83 1 e6 1 1 VEE3014 1 e4 1 1VEE3014 1 e6 1 32 VEE3000 1 e3 2 2 VEE3000 1 e4 15  70 VEE3000 1 e68914   40960 TC-83IRES 1 e6 1 1 TC-83(E181I)IRES 1 e6 1 1 VEE 3014IRES 1e6 2 36 *To calculate GMT anti-vector titers of <1:10 were arbitrarilyassigned a value of 1.

TABLE 16 Anti-VEE responses in Cynomolgus monkeys immunized with GAG-VRPVEE 2W³ 4 W 2 W 4 W 6 W 8 W 12 W 14 W 16 W 20 W replicon #¹ Rte² PP⁴ PPPB⁵ PB PB PB PB PB PB PB TC-83 1 s.c. ≦10 ≦10 80 40 40 20 10 10 10 10TC-83 2 s.c. ≦10 ≦10 ≦10 ≦10 ≦10 ≦10 20 40 20 40 TC-83 3 s.c. 10 ≦10 160320 320 320 160 160 160 80 TC-83 1 i.m. ≦10 ≦10 ≦10 ≦10 ≦10 20 ≦10 ≦10≦10 ≦10 TC-83 2 i.m. ≦10 ≦10 ≦10 10 10 40 ≦10 10 ≦10 ≦10 TC-83 3 i.m.≦10 ≦10 ≦10 10 ≦10 40 ≦10 20 ≦10 ≦10 3014 1 s.c. 640 640 20480 1024010240 5120 1280 1280 1280 1280 3014 2 s.c. 10 10 640 640 1280 640 160 8080 160 3014 3 s.c. 1280 640 10240 5120 5120 2560 1280 1280 2560 25603014 1 i.m. 160 80 40960 40960 10240 5120 5120 2560 2560 2560 3014 2i.m. 640 40 5120 10240 2560 1280 640 640 640 320 3014 3 i.m. 20 10 25605120 1280 640 640 320 320 320 ¹animal identification number ²route ofadministration: s.c. = subcutaneous; i.m. = intramuscular ³W = week ⁴PP= post-priming inoculation ⁵PB = post-first boosting inoculation

What is claimed is:
 1. A method of producing an immune response to animmunogen heterologous to an alphavirus in a subject, comprisingadministering to the subject an effective amount of a compositioncomprising infectious, propagation-defective alphavirus particles and apharmaceutically-acceptable carrier, wherein each particle comprises analphavirus replicon RNA comprising an alphavirus packaging signal andone or more heterologous RNA sequence(s) encoding an immunogenheterologous to the alphavirus, and wherein said alphavirus replicon RNAlacks sequences encoding alphavirus structural proteins, and whereineach particle comprises structural proteins from Venezuelan equineencephalitis virus TC-83.
 2. The method of claim 1, wherein thecomposition is administered via intramuscular, subcutaneous orintraperitoneal injection.
 3. The method of claim 1, wherein thealphavirus replicon RNA is from Venezuelan equine encephalitis virus. 4.The method of claim 1, wherein there is one heterologous RNA sequence orwherein there are two heterologous RNA sequences.
 5. A method ofproducing an immune response in a subject, comprising administering tothe subject an effective amount of a composition comprising infectious,propagation-defective alphavirus replicon particles and apharmaceutically-acceptable carrier, wherein each particle comprises aVenezuelan equine encephalitis (VEE) TC-83-derived alphavirus repliconRNA, said replicon RNA comprising a 5′ sequence of VEE strain TC-83 RNAwhich initiates transcription of alphavirus RNA, one or more nucleotidesequences which together encode TC-83 alphavirus nonstructural proteinsnecessary for replication of the replicon RNA, an alphavirus packagingsignal and one or more heterologous RNA sequence(s) encoding animmunogen heterologous to VEE TC-83, and a 3′ RNA polymerase recognitionsequence of VEE strain TC-83 wherein said alphavirus replicon RNA lackssequences encoding alphavirus structural proteins, and wherein eachparticle comprises structural proteins from Venezuelan equineencephalitis virus TC-83.
 6. The method of claim 5, wherein thecomposition is administered via intramuscular, subcutaneous orintraperitoneal injection.
 7. The method of claim 5, wherein there isone heterologous RNA sequence or wherein there are two heterologous RNAsequences in said replicon RNA.